Novel agonistic anti tnfr2 antibody molecules

ABSTRACT

Described are novel antagonistic antibody molecules that specifically bind to TNFR2 on a target cell and thereby block TNF-α binding to TNFR2 and block TNFR2 signaling, wherein the antibody molecules also bind to Fc receptors via the Fc region. Also described is the use of such antibody molecules in treatment of cancer or infections caused by intracellular pathogens.

FIELD OF THE INVENTION

The present invention relates to novel antagonistic antibody moleculesthat specifically bind to tumor necrosis factor receptor 2 (TNFR2) ontarget cells and thereby block the ligand TNF-α from binding to TNFR2and that also block TNFR2 signaling, which antibody molecules also bindto an Fc receptor via their Fc region. The invention also relates to usethereof in medicine, such as in treatment of cancer or infections causedby an intracellular pathogen.

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF) receptor 2 (TNFR2, TNFR-2 or TNFRII), alsoknown as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B)and CD120b, is a membrane receptor that binds tumor necrosisfactor-alpha (TNF-α or TNFα). It is found i.e. on the surface of Tcells, monocytes and macrophages, and can activate the proliferation ofTNFR2 receptor expressing cells through nuclear factor kappa B (NF-κB).Notably, TNFR2 is highly upregulated in cancer and in particular ontumor-infiltrating immune cells, e.g. regulatory T cells (Tregs), CD8⁺cytotoxic effector T cells, and different myeloid cell subpopulations.

TNFR2 has been discussed as a promising target for cancer immunotherapy,and has been described to be highly expressed on the surface of i.a.intratumoral Tregs and many human tumor cells (Williams G S et al,Oncotarget. 2016; 7(42): 68278-68291; Vanamee E S et al, Trends inMolecular Medicine, 2017, vol. 23, issue 11, 1037-1046, Frontiers inImmunology, November 2017|Volume 8|Article 1482, Sci Signal. 2018 Jan.2; 11 (511).

Regulatory T cells (which may also be called Treg cells, Tregs orT_(regs), and which formerly were known as suppressor T cells orsuppressive regulatory T cells) constitute a subpopulation of T cellscapable of suppressing other immune cells in normal and pathologicalimmune settings. Tregs are CD4 positive cells (CD4⁺ cells). There areother CD4⁺ T cells that are not Tregs; however, Tregs can be separatedfrom non-Treg CD4⁺ cells in that Tregs also are FOXP3 positive (FOXP3⁺)while the non-Treg CD4⁺ cells are FOXP3 negative (FOXP3⁻). Tregs canalso be separated from non-Treg CD4⁺ cells in that Tregs also areCD25⁺CD127^(neg/low) while the non-Treg CD4⁺ cells are eitherCD25⁻CD127⁺ or CD25⁺CD127⁺

TNFR2 has also been discussed in connection with autoimmune diseases(Faustman D L et al, Front Immunol. 2013; 4: 478, Clin TranslImmunology. 2016 Jan. 8; 5(1): J Neurosci. 2016 May 4; 36(18):5128-43)and inflammatory diseases (Ait-Ali D et al. Endocrinology. 2008 June;149(6):2840-52, Sci Rep. 2016 Sep. 7; 6:32834).

Anti-TNFR2 antibodies of different types and with variouscharacteristics have also been described previously. For example,Williams et al (Oncotarget. 2016 Oct. 18; 7(42):68278-68291) describesboth ligand blocking and ligand non-blocking agonistic antibodies.

WO 2014/124134 discloses the use of a TNFR2 agonist, such as anagonistic anti-TNFR2 antibody) and/or an NF-κB activator for in vitroproduction of a composition enriched in CD4+CD25^(hi) Tregs. Thecomposition is said to be useful in treatment of immunological disordersor infectious diseases in patients. WO 2014/124134 further disclosesTNFR2 antagonist antibodies that can bind one or two epitopes of TNFR2.The first of these epitopes included the sequence QTAQMCCSKCSPGQHAKVFCand the second epitope included one specific amino acid in one specificposition in the amino acid of human TNFRs; the second epitope mayinclude the sequence RLCAPLRKCRPGF. Such TNFR2 antagonists are said tobe useful to produce compositions enriched in lymphocytes and depletedof Tregs. In granted U.S. Pat. No. 9,821,010, originating from this PCTapplication, the antagonistic antibody has been specified as selectivelybinding to an epitope within the sequence KQEGCRLCAPLRKCRPGFGV, such asthe epitope comprising the sequence RLCAPLRKCRPGF. The TNFR2antagonists, and the compositions produced using the TNFR2 antagonists,are said to be useful in treatment of proliferative disorders, such ascancer, or infectious diseases.

WO 2016/187068 discloses antibodies capable of antagonizing tumornecrosis factor receptor superfamily members, such as TNFR2. Theantibodies are said to be useful to modulate Tregs, such as inimmunotherapy for treatment of proliferative disorders and infectiousdisease. In particular, it discloses antagonistic TNFR2 antibodiesbinding to specific epitopes of human TNFR2, and it presents a number ofspecific CDR sequences of such antibodies. Data in WO 2016/187068 issaid to demonstrate that the specific binding of the Fab regions of theantagonistic TNFR2 antibodies to TNFR2 is likely responsible formodulating Treg cell growth, rather than non-specific binding of the Fcregions of these antibodies

WO 2017/040312 discloses anti-TNFR2 antibodies, and in particularagonistic anti-TNFR2 antibodies, that are capable of promoting TNFR2signaling and having an effect on expansion or proliferation of Tregs.WO 2017/040312 discloses antibodies that bind specifically to an epitopecomprising the sequence KCSPG, but not to an epitope comprising thesequence KCRPG, thus excluding the antibodies of U.S. Pat. No. 9,821,010discussed above, or alternatively not to another TNFR superfamilymember. The agonistic antibodies are said to be useful in treatment ofimmunological diseases. WO 2017/040312 further sets out the fullsequence of human TNFR2.

WO 2017/083525 discusses pharmacological compositions comprisinganti-TNFR2 antibodies, and use thereof in treatment of disordersassociated with TNF-α and/or TNFR2, such as cancer. WO 2017/083525further discusses antibodies comprising a human IgG1 Fc domain which isnull for binding to an Fcγ receptor, and also suppression of expansionof Tregs.

WO 2017/197331 discloses antagonistic TNFR2 antibodies comprisingcomplementarity determining region-heavy chain 3 having specificsequences, and discusses reducing or inhibiting the proliferation ofTregs and/or promotion of proliferation of T effector cells.

Fc receptors are membrane proteins which are found on the cell surfaceof immune effector cells including monocytes, macrophages, dendriticcells, neutrophils, mast cells, basophils, eosinophils and NaturalKiller cells and B lymphocytes. The name is derived from their bindingspecificity for the Fc region of antibodies. Fc receptors are found onthe cell membrane—otherwise known as the plasma membrane or cytoplasmicmembrane. Fc receptors can be subdivided into activating FcγR andinhibitory FcγR, which are known to co-ordinately regulate cellularactivation through binding of aggregated immunoglobulin G Fc's, andtransmission of activating or inhibitory signals into the cell throughintracellular ITAM or ITIM motifs. FcR binding of aggregatedimmunoglobulin or immune complexes, can mediate antibody internalizationinto the cell, and can result in antibody-mediated phagocytosis,antibody-dependent cell-mediated cytotoxicity, or antigen presentationor cross-presentation. FcRs are also known to mediate or enhancecross-linking of antibody-bound cell surface receptors. Suchcross-linking is known to be required for some (Li et al. 2011.‘Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumoractivities of agonistic CD40 antibodies’, Science, 333:1030-4; White etal. 2011. ‘Interaction with FcgammaRIIB is critical for the agonisticactivity of anti-CD40 monoclonal antibody’, J Immunol, 187:1754-63) butnot all (Richman et al. 2014. ‘Anti-human CD40 monoclonal antibodytherapy is potent without FcR crosslinking’, Oncoimmunology, 3: e28610)antibodies ability to activate signaling in targeted cells, and may ormay not be required to achieve therapeutic effects.

A subgroup of the Fc receptors are Fcγ receptors (Fc-gamma receptors,FcgammaR, FcγR), which are specific for IgG antibodies. There are twotypes of Fcγ receptors: activating Fcγ receptors (also denotedactivatory Fcγ receptors) and inhibitory Fcγ receptors. The activatingand the inhibitory receptors transmit their signals via immunoreceptortyrosine-based activation motifs (ITAM) or immunoreceptor tyrosine-basedinhibitory motifs (ITIM), respectively. In humans, FcγRIIb (CD32b) is aninhibitory Fcγ receptor, while FcγRI (CD64), FcγRIIa (CD32a), Fcγ RIIc(CD32c) and FcγRIIIa (CD16a) are activating Fcγ receptors. FcγgRIIIb isa GPI-linked receptor expressed on neutrophils that lacks an ITAM motifbut through its ability to cross-link lipid rafts and engage with otherreceptors is also considered activatory. In mice, the activatingreceptors are FcγRI, FcγRIII and FcγRIV.

It is well-known that antibodies can modulate immune cell activitythrough interaction with Fcγ receptors. Specifically, how antibodyimmune complexes modulate immune cell activation is determined by theirrelative engagement of activating and inhibitory Fcγ receptors.Different antibody isotypes bind with different affinity to activatingand inhibitory Fcγ receptors, resulting in different A:I ratios(activation:inhibition ratios) (Nimmerjahn et al; Science. 2005 Dec. 2;310(5753): 1510-2).

By binding to an inhibitory Fcγ receptor, an antibody can inhibit, blockand/or down-modulate effector cell functions. By binding to aninhibitory FcγR, antibodies can further stimulate cell activationthrough aggregation of antibody-targeted signaling receptors on a targetcell (Li et al. 2011. ‘Inhibitory Fcgamma receptor engagement drivesadjuvant and anti-tumor activities of agonistic CD40 antibodies’,Science, 333:1030-4; White et al. 2011. ‘Interaction with FcgammaRIIB iscritical for the agonistic activity of anti-CD40 monoclonal antibody’, JImmunol, 187:1754-63; White et al. 2014. ‘Fcgamma receptor dependency ofagonistic CD40 antibody in lymphoma therapy can be overcome throughantibody multimerization’, J Immunol, 193: 1828-35).

By binding to an activating Fcγ receptor, an antibody can activateeffector cell functions and thereby trigger mechanisms such asantibody-dependent cellular cytotoxicity (ADCC), antibody dependentcellular phagocytosis (ADCP), cytokine release, and/or antibodydependent endocytosis, as well as NETosis (i.e. activation and releaseof NETs, Neutrophil extracellular traps) in the case of neutrophils.Antibody binding to an activating Fcγ receptor can also lead to anincrease in certain activation markers, such as CD40, MHCII, CD38, CD80and/or CD86.

Recent data published by i.a. the inventors demonstrate a critical anddifferential dependence of CD8 T cell agonist and Treg-depletinganti-4-1 BB antibodies for binding to activating and inhibitory FcγRsrespectively, for therapeutic efficacy (Buchan et al., ‘Antibodies toCostimulatory Receptor 4-1 BB Enhance Anti-tumor Immunity via TRegulatory Cell Depletion and Promotion of CD8 T Cell EffectorFunction’, Immunity 2018 49(5):958-970). Moreover, and critically,simultaneous administration of CD8 T cell agonist and Treg depletinganti-4-1 BB antibodies optimized for binding to activating andinhibitory FcγR respectively, impaired therapeutic activity. These datademonstrate the critical importance of developing antibodies withappropriate and tailored engagement of activating and inhibitory FcγRsto maximize therapeutic activity of antibodies with distinctmechanism-of-action. At the same time, they demonstrate that suboptimalengagement of activating and inhibitory FcγRs may severely reducetherapeutic efficacy.

These data were surprising as they contrasted with findings forantibodies to other TNFSR members, notably immune stimulatory anti-CD40antibodies, which show an obligate need for engagement of theinhibitory, but not activating, FcγRs (Li et al. 2011. ‘InhibitoryFcgamma receptor engagement drives adjuvant and anti-tumor activities ofagonistic CD40 antibodies’, Science, 333:1030-4; White et al. 2011.‘Interaction with FcgammaRIIB is critical for the agonistic activity ofanti-CD40 monoclonal antibody’. J Immunol, 187:1754-63). Taken together,these results demonstrate that FcγR-dependence can vary betweenantibodies to different targets of the same receptor superfamily, andeven between different types of antibodies to the same target in amanner that is not easily predictable yet may be critical to understandand harness when developing antibodies for therapeutic use.

SUMMARY OF THE INVENTION

In the work leading to the present invention, and also to a parallelinvention, two major different groups of anti-TNFR2 antibodies withpowerful therapeutic activity, and different characteristics andmechanism-of-actions were identified.

The inventors first identified a powerful therapeutic activity ofantagonistic anti-TNFR2 antibodies that block TNF-α binding to TNFR2receptor. The activity of such antibodies was shown to be dependent onFcγR-interactions, and in particular binding to activatory FcγR, for invivo therapeutic activity. This group or category of powerful anti-TNFR2therapeutic reagents was found to be characterized by 1) pronouncedblock and inhibition of TNF-α (the ligand) induced TNFR2-signalling, and2) an activity dependent on FcγR-engagement, benefitting most stronglyfrom engaging activating over inhibitory FcγRs.

The inventors then identified a distinct group of anti-TNFR2 antibodieswith equally powerful therapeutic activity in vivo, but whosecharacteristics in many respects are opposite to those of theantagonistic, blocking type of TNFR2 antibodies constituting the firstgroup and the present invention. The anti-TNFR2 antibodies of thissecond group do not depend on TNF-α blockade or inhibition ofTNFR2-signalling for therapeutic activity, but rather is characterizedby strong activation of TNFR2-signalling. Further contrasting with theblocking antibodies of the first group, the agonistic antibodies of thesecond group do not show obligate dependence onantibody:FcγR-engagement, even though their activity is improved withFcγR:engaging antibody variants. In further contrast to the antagonisticand blocking antibodies of the first group, the agonistic antibodies ofthe second group show greatest activity in antibody variants withimproved binding to inhibitory vs activating FcγR.

The present invention relates to the first group of anti-TNFR2antibodies, i.e. to antagonistic antibody molecules that by bindingspecifically to TNFR2, block TNF-α from binding to TNFR2 and also blockTNFR2 signaling. These antibody molecules also have an Fc region thatbinds to an Fc receptor, which is useful in conferring FcγR-dependentelimination or functional modulation of TNFR2 positive cells, such ase.g. Treg depletion or modulation of tumor associated macrophages.

Agonistic antibodies belonging to the second group are used in theExamples below for comparison with the antagonistic, blocking TNFR2antibody molecules of the present invention. In the examples, also otherantibodies with some characteristics similar to either those of thefirst or second group, or both, are used for comparison, as furtherexplained below.

Thus, the present invention relates to antagonistic antibody moleculesthat specifically bind to TNFR2 on a target cell and thereby block TNF-αbinding to TNFR2 and block TNFR2 signaling, and wherein the antibodymolecules also bind to an Fcγ receptor via their Fc region.

The present invention also relates to specific examples of such novelantagonistic and blocking anti-TNFR2 antibody molecules.

The present invention also relates to isolated nucleotide sequencesencoding at least one of the above antibody molecules.

The present invention also relates to plasmids comprising at least oneof the above nucleotide sequences.

The present invention also relates to viruses comprising at least one ofthe above nucleotide sequences or plasmids.

The present invention also relates to cells comprising one of the abovenucleotide sequence, or one of the above plasmids, or one of the aboveviruses.

The present invention also relates to the above antibody molecules,nucleotide sequences, plasmids, viruses and/or cells for use inmedicine.

The present invention also relates to the above antibody molecules,nucleotide sequences, plasmids, a viruses and/or cells for use in thetreatment of cancer or infections caused by an intracellular pathogen.

The present invention also relates to the above antibody molecules,nucleotide sequences, plasmids, viruses and/or cells for use in thetreatment of cancer or infections caused by an intracellular pathogen.

The present invention also relates to pharmaceutical compositionscomprising or consisting of at least one of the above antibodymolecules, nucleotide sequences, plasmids, viruses and/or cells, andoptionally a pharmaceutically acceptable diluent, carrier, vehicleand/or excipient. Such a pharmaceutical composition may be used in thetreatment of cancer or infections caused by an intracellular pathogen.

The present invention also relates to methods for treatment of cancer orinfections caused by an intracellular pathogen in a patient comprisingadministering to the patient a therapeutically effective amount of atleast one of the above antibody molecule, nucleotide sequences,plasmids, viruses and/or cells.

The present invention also relates to antibody molecules, antibodymolecules for use, isolated nucleotide sequences, isolated nucleotidesequences for use, plasmids, plasmids for use, viruses, viruses for use,cells, cells for use, uses, pharmaceutical compositions and methods oftreatment as described herein with reference to the accompanyingdescription, examples and/or figures.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention relates to antagonistic antibody moleculesthat specifically bind to TNFR2 on a target cell and thereby block TNF-αbinding to TNFR2 and block TNFR2 signaling, and wherein the antibodymolecules also bind to an Fcγ receptor via their Fc region.

The antagonistic antibody molecules disclosed herein block both TNF-αfrom binding to TNFR2 and TNFR2 signaling. That the antagonisticantibody molecules block TNF-α from binding to TNFR2 means herein thatan antibody molecule that binds to the receptor TNFR2 thus prevents theligand TNF-α from binding to the same receptor. This is demonstrated inmore detail in Example 3. That the antagonistic antibody moleculesdisclosed herein block TNFR2 signaling means that they block TNFR2mediated cell activation. It has been clearly demonstrated that TNF-αmediated signaling through TNFR2 starts a signaling cascade that ends inactivation of the nuclear transcription factor NFκB (Thommesen et al.“Distinct differences between TNF receptor 1- and TNF receptor2-mediated activation of NFkappaB”. J Biochem Mol Biol. 2005 May 31;38(3):281-9; Yang et al. “Role of TNF-TNF Receptor 2 Signal inRegulatory T Cells and Its Therapeutic Implications”. Front Immunol.2018 Apr. 19; 9:784). This in turn results in activation of the cell andsynthesis of several pro-inflammatory factors, one of them being IFN-γin NK cells (Liu et al. “NF-κB signaling in inflammation”. SignalTransduct Target Ther. 2017; 2. pii: 17023; Tato et al. “Opposing rolesof NF-kappaB family members in the regulation of NK cell proliferationand production of IFN-gamma”. Int Immunol. 2006 April; 18(4):505-13).Herein the terms TNFR2 signaling and TNFR2 activation are usedinterchangeably.

The antibody molecules bind specifically to TNFR2. It is well known thatan antibody specifically binds to or interacts with a defined targetmolecule or antigen, and that this means that the antibodypreferentially and selectively binds its target and not a molecule whichis not a target. By “antibody molecule that specifically binds TNFR2” or“TNFR2 specific antibody molecule” we mean an antibody that binds TNFR2protein in a dose-dependent manner but not to an unrelated protein. Inaddition, the same antibody binds cells that endogenously express TNFR2,and this binding can be blocked out by pre-incubation of the same cellswith a commercially available polyclonal TNFR2 antibody reagent, showingthat no non-specific binding can be detected when TNFR2 is masked by apolyclonal reagent. This is shown in example 2.

The antibody molecule that specifically binds TNFR2 (or the anti-TNFR2antibody molecule) refers to an antibody molecule that specificallybinds to at least one epitope in the extracellular domain of TNFR2. Cellsurface antigen and epitope are terms that would be readily understoodby one skilled in immunology or cell biology.

Methods of assessing protein binding are known to the person skilled inbiochemistry and immunology. It would be appreciated by the skilledperson that those methods could be used to assess binding of an antibodyto a target and/or binding of the Fc region of an antibody to an Fcreceptor; as well as the relative strength, or the specificity, or theinhibition, or prevention, or reduction in those interactions. Examplesof methods that may be used to assess protein binding are, for example,immunoassays, BIAcore, western blots, radioimmunoassay (RIA) andenzyme-linked immunosorbent assays (ELISAs) and Flow cytometry (FACS)(See Fundamental Immunology Second Edition, Raven Press, New York atpages 332-336 (1989) for a discussion regarding antibody specificity).

The target cells expressing the TNFR2 to which the blocking antibodybinds in accordance with the present invention include immune cellsand/or tumor cells, as discussed above and below. The effect of thebinding of the antagonistic antibody molecules according to theinvention to TNFR2 may be a change in composition of the cells indiseased tissue. This change in composition may occur through a changein number and/or frequency of TNFR2-expressing cells in the diseasedtissue. For example, the effect in cancer include increased intratumoralT cell numbers, increased ratio of CD8⁺ T cells/Treg (i.e. the number ofCD8⁺ T cells to the number of Tregs), and/or increased numbers ofmyeloid cells associated with anti-tumor as opposed to pro-tumorcharacteristics. This is demonstrated in Example 5. In some embodiments,these effects result in reduced numbers of tissue Tregs, increasednumbers of CD8⁺ effector T cells, and altered composition of tissuemyeloid cell subsets. The modulation of TNFR2-expressing cells in tissuefollowing in vivo therapeutic invention with a surrogate (3-F10) to theantagonistic antibody molecules according to the invention is showndetail in Example 5. To test for similar effects of human antagonisticantibody molecules according to the invention, an analogous experimentcan be performed in mice that have been deleted in mouse TNFR2 and madetransgenic for human TNFR2. Alternatively, and preferably, althoughrequiring significant time and resource, animals transgenic for humanTNFR2 and human FcγR can be generated in analogous manner compared tothat described for CD40 and hFcγR previously (Dahan et al. 2016.‘Therapeutic Activity of Agonistic, Human Anti-CD40 MonoclonalAntibodies Requires Selective FcgammaR Engagement’, Cancer Cell, 29:820-31). Such humanized TNFR2 FcγR mice can then be used analogously totest for similar effects of human antagonistic antibody moleculesaccording to the invention.

Diseased tissue means in this context either tumor tissue (i.e. allcells in the tumor microenvironment including tumor cells, immune cells,endothelial cells and stromal cells) or tissue infected by anintracellular pathogen.

To decide whether or not an antibody molecule blocks ligand binding toTNFR2, it is possible to use an ELISA assay determining the amount ofbound TNF-α ligand to immobilized TNFR2 receptor in the presence ofTNFR2 specific antibodies. A blocking antibody will prevent the ligand,TNF-α, from binding to the immobilized receptor TNFR2. This isdemonstrated and explained in more detail in Example 3 below.

A blocking antibody molecule according to the invention is a completeblocker, which additionally is capable of antagonizing TNFR2 signaling.

A complete blocker is defined herein as an antibody molecule thatreduces the TNF-α binding to TNFR2 by more than 98%, i.e. up to 100%,compared to TNF-α binding in the presence of only an isotype controlantibody molecule. An isotype control antibody is an antibody raisedtowards a protein or other structure that is not present in any form inthe assay under study. The isotype control ideally has the sameframework but at least the same Fc part as the comparing antibodies.This is well known to the skilled person. In the examples describedherein, the isotype control had the same framework, the same Fc part,and was specific for Fluorescein isothiocyanate (FITC). In someembodiments the complete blocker reduces the TNF-α binding with morethan 99.5%.

Other types of blockers are partial blockers and weak blockers. As usedherein, a partial blocker is an antibody molecule that reduces the TNF-αbinding to TNFR2 by 60-98% (e.g. by 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98% and all decimalnumbers in between) compared to TNF-α binding in the presence of only anisotype control antibody molecule, and a weak blocker is an antibodymolecule that reduces the TNF-α binding to TNFR2 by less than 60%, suchas 50-59.9% (or 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 59.9% and alldecimal numbers in between), compared to TNF-α binding in the presenceof only an isotype control antibody molecule.

On the contrary, a non-blocking TNFR2 antibody molecule is an antibodymolecule that reduces the TNF-α binding to TNFR2 by less than 50%compared to TNF-α binding in the presence of only an isotype controlantibody molecule. In some embodiments, this is determined in high-dose,one-point ELISA as or a dose-titration ELISA as shown in Example 3 andFIGS. 6 and 7.

Partially blocking, weak blocking and non-blocking antibodies are usedin the examples for comparison with the antagonistic blocking antibodymolecules of the present invention.

Several properties and features can underlie and (co-)determine thebiological activity of antibodies. Besides ability to block ligand frombinding to receptor, important such properties include antibody abilityto modulate receptor signaling i.e. agonize or antagonize receptorsignaling, and antibody dependence on FcγR interactions to confertherapeutic activity.

We first characterized the ability of complete blocking, partialblocking and non-blocking antibodies to modulate TNFR2 signaling. Twoextremes were identified.

On the first extreme, we identified antibodies that completely blockedligand-binding to TNFR2, which blocked TNF-α induced TNFR2 signaling,and which did not themselves induce signaling upon binding tocell-endogenously expressed TNFR2. This group of ligand-blocking,antagonistic, antibodies forms the basis for the present invention.

On the other extreme, we identified antibodies that do not blockligand-binding to TNFR2, but upon binding to TNFR2 endogenouslyexpressing cells agonized the receptor. This second group of antibodiesconstitute a separate invention and are included herein for comparison.

Antibodies and categories defined by partial blocking agonistic, partialblocking non-agonistic, and complete blocking non-antagonistic, wereadditionally identified demonstrating the complex biology and greatheterogeneity of anti-TNFR2 antibodies clearly demonstrating that theantibodies of the present invention forms a unique group.

To determine whether an antibody has agonist or antagonistic activity itis possible to use a Natural Killer (NK) cell assay as described inExample 4. Briefly, NK cells have been described to respond to IL-2 andIL-12 stimuli with secretion of IFN-γ. Soluble TNF-α is endogenouslyproduced and present at robust but suboptimal concentrations (˜20-100pg/ml), for TNFR2 signaling, meaning that IFN-γ can be both increasedand decreased through modulation of TNFR2 signaling. Consequently,exogenous addition of TNF-α at TNFR2 signaling optimal concentrationenhances IFN-γ concentrations in this assay, as does incubation withagonist anti-TNFR2 antibody (FIG. 8 C). Contrarily, co-incubation withanti-TNF-α antibody or herein described ligand-blocking antagonistantibodies, decreases IFN-γ release in this assay. Thus, this assay canbe used to identify agonist or antagonist activity, or lack thereof, ofanti-TNFR2 antibodies. (TNFα Augments Cytokine-Induced NK Cell IFNγProduction through TNFR2. Almishri W. et al. J Innate Immun. 2016;8:617-629) Consequently, in this experimental set-up, an antagonisticantibody prevents TNF-α induced signaling in TNFR2-expressing cells, anddoes not itself stimulate the TNFR2 receptor upon binding to the same.Specifically, antagonistic antibodies in this assay do not increaseIFN-γ release upon binding to above mentioned NK cells but ratherinhibit IFN-γ release. As shown in FIG. 8, complete blocking antibodiesdescribed in this invention did not induce TNFR2 signaling, but ratherreduced TNFR2 signaling in this TNF-α containing NK cell assay,resulting in lower amounts of released IFN-γ. As indicated in FIG. 8Example 4, antibodies of this invention can therefore be classified asligand-blocking antagonistic anti-TNFR2 antibodies. Using this assay, anantagonistic antibody is defined as an antibody resulting in >30% (e.g.35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%) reduction ofIFN-γ release given that the basal TNF-α levels in the culture is atleast 20 pg/ml. Since this assay uses primary cells from PBMC donors, atleast 4 donors needs to be included and the mean values should becalculated from all donors. Cells from each donor to be included incalculation of means must have responded to the positive control(soluble TNF-α) treatment with >100% (>2-fold) increased IFN-γ levelsrelative to treatment with isotype control.

The antagonist activity can also be demonstrated using IL-2 mediatedactivation of memory T cells. Activation is here measured byupregulation of the T cell activation marker CD25. Using this assay,addition of non-blocking agonistic TNFR2 antibodies further upregulateCD25 expression whereas the blocking antagonistic TNFR2 antibodiesaccording to the invention result in lower CD25 expression compared toisotype control. This is true for the human antibodies of the inventionas well as murine surrogate antibodies and is shown in example 4.

In addition to binding to TNFR2, and thereby blocking TNF-α binding andsignaling, the antibody molecules according to the invention also bindto Fcγ receptors. An obligate dependence on FcγR interactions ofanti-TNFR2 antibodies belonging to the TNF-α-blocking antagonistic groupof antibodies of the present invention for therapeutic efficacy, wasdemonstrated in mouse cancer experimental models using antibody variantswhich productively engage, or do not productively engage, FcγR binding.The obligate dependence of such ligand blocking antagonistic antibodiesfor in vivo therapeutic activity, and their preferentialbinding/engagement of activatory over inhibitory FcγRs for maximaltherapeutic in vivo activity, is shown in Example 5. Data demonstratingthe FcγR-independent in vivo activity of agonist, non-blockinganti-TNFR2 antibodies, and their differential preferential engagement ofinhibitory over activating FcγRs for maximal therapeutic activity isincluded in this example for comparative and contrasting purposes only.Collectively our data demonstrate that several types of anti-TNFR2antibodies can be generated. Further, our data demonstrate that it isnot trivial, and could not be predicted, which antibody variants wouldbe therapeutically most efficacious, whether they would rely onblocking, agonist or antagonist (extrinsic or intrinsic) properties, andwhether or not they would depend on FcγR-engagement, or be mostefficacious in antibody formats associated with preferential/strongbinding to activating or inhibitory Fc gamma receptors.

The relatively high homology between mouse and human FcγR systemsaccounts for many of the general aspects of conserved FcγR-mediatedmechanisms between the species. However, mouse and human IgG subclassesdiffer in their affinities for their cognate FcγRs, making it importantwhen translating FcγR-mediated observations in the mouse system intohuman IgG-based therapeutics to choose an antibody, antibody subclassand/or engineered subclass variant, that shows appropriate binding tohuman activating vs inhibitory FcγRs. The affinity and/or avidity ofhuman antibody molecules for individual human Fcγ receptors can bedetermined using surface plasmon resonance (SPR). In some embodiments,the blocking TNFR2 antibody molecule binds with higher affinity toactivating Fcγ receptors than to inhibitory Fcγ receptors. With higheraffinity to activating Fcγ receptors than to inhibitory Fcγ receptors,we include the meaning of variants that bind with higher affinity toactivating Fcγ receptors, e g. FcγRIIA, FcγRIIIA and/or FcγRI, comparedwith the inhibitory Fcγ receptor.

In some embodiments, the antibody molecule is an IgG, which may bind toFcγ receptors through normal interaction between the Fc region of theantibody molecule and the Fcγ receptor.

In some embodiments, the antagonistic, blocking TNFR2 antibody moleculeis a human IgG1. It is well known that human IgG1 binds with highaffinity to activating human FcγRI, and with lower and similar affinityto human activatory Fcγ receptors FcγRIIA, FcγRIIIA, and to humaninhibitory FcγRIIB. This has been demonstrated e.g. using surfaceplasmon resonance (SPR).

In some embodiments, the antagonistic, blocking TNFR2 antibody moleculeis an IgG antibody molecule showing improved binding to one or severalactivatory Fc receptors and/or being engineered for improved binding toone or several activatory Fcγ receptors and/or being engineered forimproved relative binding to activatory over inhibitory Fcγ receptors.In some embodiments, the anti-TNFR2 antibody is an Fc-engineered humanIgG1 antibody. Examples of such engineered antibody variants includeafucosylated antibodies with selective improved antibody binding toFcγRIIIA, and antibodies engineered by directed, mutational, or by othermeans, amino acid substitution resulting in improved binding to one orseveral activating Fcγ receptors compared to inhibitory FcγRIIB(Richards et al. 2008. ‘Optimization of antibody binding to FcgammaRIIaenhances macrophage phagocytosis of tumor cells’, Mol Cancer Ther, 7:2517-27; Lazar et al. 2006. ‘Engineered antibody Fc variants withenhanced effector function’, Proc Natl Acad Sci USA, 103: 4005-10)

In some embodiments, the human IgG antibody that is engineered forimproved binding to activating Fc gamma receptors may be a human IgGantibody carrying the two mutations S239D and I332E, or the threemutations S239D, I332E and A330L, and/or G236A mutations in its Fcportion. In some embodiments, the human IgG antibody that is engineeredfor improved binding to activating Fc gamma receptors may be anafucosylated human IgG antibody.

The Fcγ receptor to which the Fc region of the antagonistic, blockingantibody molecules of the present invention binds can be an Fcγ receptorexpressing immune effector cell as described above.

The binding of the TNFR2 specific antibody molecule to TNFR2 surfacereceptors on target cell, and co-engagement of FcγR on the same cell orimmune effector cells in close proximity may result in depletion, orfunctional modulation, of the TNFR2 positive target cells to which theantibody molecule binds. By depletion of a cell, we refer herein todepletion, deletion or elimination of the cell through physicalclearance of cells.

Depletion of cells may be achieved through ADCC, i.e. antibody-dependentcell-mediated cytotoxicity or antibody-dependent cellular cytotoxicity,and/or ADCP, i.e. antibody dependent cellular phagocytosis. This meansthat when an antibody molecule as described herein is administered to apatient, such as a human, it binds specifically to TNFR2 expressed onthe surface of cells, such as Tregs, and this binding results indepletion of the cells. In general, high-expressing cells are deletedmore efficaciously compared to low-expressing cells. As shown in example5, FIG. 14, Tregs are the highest expressing cells in the tumor setting.

ADCC is an immune mechanism through which Fc receptor-bearing effectorcells can recognize and kill—i.e. deplete—antibody-coated target cellsexpressing tumor-derived antigens, i.e. in the present case, TNFR2, ontheir surface. ADCP is a similar mechanism, although it results in thetarget cells being killed—i.e. depleted—through phagocytosis instead ofcytotoxicity.

In addition, improved binding of the Fc region of the antibody moleculesto the Fcγ receptor may also improve the depletion of the target cellthrough Fc receptor dependent death via ADCC or ADCP. This is inparticular relevant for antibody molecules with improved binding toactivating Fcγ receptors.

That the antibody molecules have a depleting effect on TNFR2 positivecells means that upon administration to a patient, such as a human, suchan antibody molecule binds specifically to TNFR2 expressed on thesurface of TNFR2 positive cells, and this binding results in depletionof such target cells.

The cells that are depleted can be a number of different cells, asexplained above in connection with the discussion of what the targetcell is. It is in general the cells that have the highest expression ofTNFR2 that will be depleted. Other cells that also express TNFR2 but notas high may also be depleted, but to a minor extent compared to thecells that have the highest TNFR2 expression.

As mentioned above, TNFR2 is highly expressed on Tregs found in tumorsin various cancer patients, and in such patients the antibody moleculeof the invention will preferentially bind to Tregs and thus result indepletion of Tregs. Tregs have an inhibiting effect on theproliferation, activation and cytotoxic capacity of other immune cellssuch as CD8 positive (CD8⁺) cells, and therefore depletion of Tregswill, at least indirectly, result in increased proliferation, activationand possibly migration of CD8⁺ cells and thus an increase of the numberof intratumoral CD8⁺ cells. CD8⁺ T cells are essential for an immunemediated clearance of tumor cells (McKinney et al. Curr Opin Immunol.2016 December; 43:74-80; Klebanoff et al. Immunol Rev. 2006 June;211:214-24; Alexander-Miller. Immunol Res. 2005; 31(1):13-24). Hence, anincrease in proliferation, activation and cytotoxic capacity of CD8⁺ Tcells would be highly beneficial for a cancer patient and can lead tocancer eradication.

An increase in proliferation, activation and cytotoxic capacity of CD8⁺T cells would also be highly beneficial for treatment of an infectioncaused by an intracellular pathogen. Antigens from intracellularpathogens are typically presented on MHC I molecules which areinstrumental in activating CD8⁺ T cells. CD8⁺ T cells thereby recognizeinfected cells and lyse them, thereby destroying the pathogen.

The binding of the TNFR2 specific antibody molecule and blocking ofTNF-α signaling may also result in functional modulation of cellphenotypes, e.g. modulation of a pro-tumoral myeloid cell into a myeloidcell with tumoricidal properties.

In some embodiments, the TNFR2 positive cells, i.e. the target cells,are CD4 positive (CD4⁺) cells, i.e. cells that express CD4.

In some embodiments, the TNFR2 positive cells are both CD4⁺ and FOXP3⁺,i.e. expressing both CD4 and FOXP3. These cells are Tregs. CD8⁺ T cellsalso express TNFR2, but Tregs express significantly higher levels ofTNFR2 than CD8 positive T cells, as shown in FIG. 14 in example 5. Thismakes Tregs more susceptible to depletion compared to lower expressingCD8⁺ cells.

In some situations, the TNFR2 is preferentially expressed on immunecells in the tumor microenvironment (tumor infiltrating cells, TILS).

In some embodiments, the Tregs will be the cells in a tumormicroenvironment that have the highest expression of TNFR2, resulting inthe antibody molecules that specifically bind to TNFR2 (or theanti-TNFR2 antibody molecules) having a Treg depleting effect. This isdiscussed in more detail below, e.g. in Example 5 and in connection withFIGS. 13 and 15.

In some embodiments, TNFR2 positive cells will be Tregs in a solidtumor. Such Tregs will have very high expression of TNFR2, and thereforeadministration of an antibody molecules that specifically binds toTNFR2, will preferentially result in depletion of such Tregs.

To decide whether an antibody molecule is an antibody molecule that hasa depleting effect on TNFR2 positive cells as referred to herein, it ispossible to use an in vivo test in a PBMC-NOG/SCID model. This in vivotest is based on the combined use of PBMC mice and NOG/SCID mice, whichis herein called a PBMC-NOG/SCID model. Both NOG mice and SCID mice areknown to the skilled person (Ito M et al, (2002) NOD/SCID/γc^(null)mouse: an excellent recipient mouse model for engraftment of humancells. Blood 100(9):3175-3182; Bosma G C et al; Nature. 1983 Feb. 10;301(5900):527-30; A severe combined immunodeficiency mutation in themouse) and also the PBMC-NOG model is known (Cox et al.“Antibody-mediated targeting of the Orai1 calcium channel inhibits Tcell function”. PLoS One. 2013 Dec. 23; 8(12):e82944; and Søndergaard etal. “Human T cells depend on functional calcineurin, tumor necrosisfactor-α and CD80/CD86 for expansion and activation in mice.” Clin ExpImmunol. 2013 May; 172(2):300-10.) The in vivo test in the PBMC-NOG/SCIDmodel comprises the following consecutive nine steps:

1) Human PBMCs (peripheral blood mononuclear cells) are isolated, washedand resuspended in sterile PBS. In some embodiments, the PBMCs areresuspended in PBS at 75×10⁶ cells/ml.

2) NOG mice are injected i.v. (intravenously) with an appropriateamount, such as 200 μl, of the cell suspension from step 1). If 200 μlare injected, this corresponds to 15×10⁶ cells/mouse.

3) A suitable time, such as 2 weeks, after injection, the spleens fromthe NOG mice are isolated and rendered into a single cell suspension.Optionally, a small sample from the single cell suspension is taken todetermine the expression of TNFR2 on human T cells by FACS, in order toconfirm the TNFR2 expression.

4) The cell suspension from step 3) is resuspended in sterile PBS. Insome embodiments, the cell suspension is resuspended in sterile PBS at50×10⁶ cells/ml. If the optional TNFR2 expression determination isincluded in step 3, the rest of the cell suspension is then resuspendedin step 4.

5) SCID mice are injected i.p. (intraperitoneally) with an appropriateamount, such as 200 μl, of the suspension from step 4. If 200 μl areinjected, this corresponds to 10×10⁶ cells/mouse.

6) A suitable time, such as 1 hour, after the injection in step 5) theSCID mice are treated with an appropriate amount, such as 10 mg/kg, ofeither the antibody molecule to be tested, a positive control antibody(e.g. anti-CD25 antibodies known to deplete Tregs) or an isotype controlmonoclonal antibody.

7) The intraperitoneal fluid of the treated SCID mice is collected asuitable time, such as 24 hours, after the treatment in step 6).

8) Human T cell subsets are identified and quantified by FACS usingfollowing markers: CD45, CD4, CD8, CD25 and/or CD127. It is wellestablished that human Tregs can be distinguished in a human PBMCpopulation as being CD4⁺CD25⁺CD127^(low/−).

9) The results from identification and quantification of the T ceilsubsets from the mice treated with the tested antibody molecule iscompared to the results from identification and quantification of the Tcell subsets from the mice treated with a positive control antibody andto the results from identification and quantification of the T cellsubsets from the mice treated with isotype control monoclonal antibody.A lower number of Tregs in the intraperitoneal fluid from mice treatedwith the antibody molecule to be tested compared to the number of Tregsin the intraperitoneal fluid from mice treated with isotype controldemonstrates that the antibody molecule has a depleting effect on TNFR2positive Tregs

This assay is demonstrated in more detail below in Example 5, incombination with FIG. 15.

As mentioned above, other cells can also express TNFR2, such as cancercells. In some embodiments, the antibody molecule binds preferentiallyto TNFR2 expressed on cancer cells, and the binding then results indepletion of the cancer cells directly.

Antibodies are well known to those skilled in the art of immunology andmolecular biology. Typically, an antibody comprises two heavy (H) chainsand two light (L) chains. Herein, we sometimes refer to this completeantibody molecule as a full-size or full-length antibody. The antibody'sheavy chain comprises one variable domain (VH) and three constantdomains (CH1, CH2 and CH3), and the antibody's molecule light chaincomprises one variable domain (VL) and one constant domain (CL). Thevariable domains (sometimes collectively referred to as the F_(v)region) bind to the antibody's target, or antigen. Each variable domaincomprises three loops, referred to as complementary determining regions(CDRs), which are responsible for target binding. The constant domainsare not involved directly in binding an antibody to an antigen, butexhibit various effector functions. Depending on the amino acid sequenceof the constant region of their heavy chains, antibodies orimmunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and inhumans several of these are further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2.

Another part of an antibody is the Fc region (otherwise known as thefragment crystallizable domain), which comprises two of the constantdomains of each of the antibody's heavy chains. As mentioned above, theFc region is responsible for interactions between the antibody and Fcreceptor.

The term antibody molecule, as used herein, encompasses full-length orfull-size antibodies as well as functional fragments of full lengthantibodies and derivatives of such antibody molecules.

Functional fragments of a full-size antibody have the same antigenbinding characteristics as the corresponding full-size antibody andinclude either the same variable domains (i.e. the VH and VL sequences)and/or the same CDR sequences as the corresponding full-size antibody. Afunctional fragment does not always contain all six CDRs of acorresponding full-size antibody. It is appreciated that moleculescontaining three or fewer CDR regions (in some cases, even just a singleCDR or a part thereof) are capable of retaining the antigen-bindingactivity of the antibody from which the CDR(s) are derived. For example,in Gao et al, 1994, J. Biol. Chem., 269: 32389-93 it is described that awhole VL chain (including all three CDRs) has a high affinity for itssubstrate.

Molecules containing two CDR regions are described, for example, byVaughan & Sollazzo 2001, Combinatorial Chemistry & High ThroughputScreening, 4: 417-430. On page 418 (right column—3 Our Strategy forDesign) a minibody including only the H1 and H2 CDR hypervariableregions interspersed within framework regions is described. The minibodyis described as being capable of binding to a target. Pessi etai, 1993,Nature, 362: 367-9 and Bianchi et al., 1994, J. Mol. Biol., 236: 649-59are referenced by Vaughan & Sollazzo and describe the H1 and H2 minibodyand its properties in more detail. In Qiu et al., 2007, NatureBiotechnology, 25:921-9 it is demonstrated that a molecule consisting oftwo linked CDRs are capable of binding antigen. Quiocho 1993, Nature,362: 293-4 provides a summary of “minibody” technology. Ladner 2007,Nature Biotechnology, 25:875-7 comments that molecules containing twoCDRs are capable of retaining antigen-binding activity.

Antibody molecules containing a single CDR region are described, forexample, in Laune et al, 1997, JBC, 272: 30937-44, in which it isdemonstrated that a range of hexapeptides derived from a CDR displayantigen-binding activity and it is noted that synthetic peptides of acomplete, single, CDR display strong binding activity. In Monnet et al.,1999, JBC, 274: 3789-96 it is shown that a range of 12-mer peptides andassociated framework regions have antigen-binding activity and it iscommented on that a CDR3-like peptide alone is capable of bindingantigen. In Heap et ai, 2005, J. Gen. Virol., 86:1791-1800 it isreported that a “micro-antibody” (a molecule containing a single CDR) iscapable of binding antigen and it is shown that a cyclic peptide from ananti-HIV antibody has antigen-binding activity and function. In Nicaiseet al, 2004, Protein Science, 13:1882-91 it is shown that a single CDRcan confer antigen-binding activity and affinity for its lysozymeantigen.

Thus, antibody molecules having five, four, three or fewer CDRs arecapable of retaining the antigen binding properties of the full-lengthantibodies from which they are derived.

The antibody molecule may also be a derivative of a full-lengthantibody, or a fragment of such an antibody provided that such aderivative or fragment retains the Fcγ receptor binding capability. Whena derivative is used, it should have the same antigen bindingcharacteristics as the corresponding full-length antibody in the sensethat it binds to the same epitope on the target as the full-lengthantibody.

Thus, by the term “antibody molecule”, as used herein, we include alltypes of antibody molecules and functional fragments thereof andderivatives thereof, including: monoclonal antibodies, polyclonalantibodies, synthetic antibodies, recombinantly produced antibodies,multi-specific antibodies, bi-specific antibodies, human antibodies,antibodies of human origin, humanized antibodies, chimeric antibodies,single chain antibodies, antibody heavy chains, homo-dimers of antibodyheavy chains, heterodimers of antibody heavy chains, and heterodimers ofantibody light chains.

Further, the term “antibody molecule”, as used herein, includes allclasses of antibody molecules and functional fragments, including: IgG,IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE, unless otherwisespecified.

In some embodiments, the antibody molecule is a human antibody molecule,a humanized antibody molecule or an antibody molecule of human origin.In some such embodiments, the antibody molecule is an IgG antibody. Insome embodiments, the antibody molecule is of an isotype that engagesactivating Fc receptors in an optimal way. In some embodiments theantibody molecule is an IgG1 antibody.

The skilled person will appreciate that the mouse IgG2a and human IgG1engage with activatory Fcγ receptors, and share the ability to activatedeletion of target cells through activation of activatory Fcγ receptorbearing immune cells by e.g. ADCP and ADCC. In some embodiments, theanti-TNFR2 antibody is a murine or a humanized murine IgG2a antibody.

In some embodiments, the antibody molecule that specifically binds TNFR2is human IgG2 antibody molecule.

In some embodiments, the anti-TNFR2 antibody is a murine antibody thatis cross-reactive with human TNFR2.

As outlined above, different types and forms of antibody molecules areencompassed by the invention, and would be known to the person skilledin immunology. It is well known that antibodies used for therapeuticpurposes are often modified with additional components which modify theproperties of the antibody molecule.

Accordingly, we include that an antibody molecule described herein or anantibody molecule used as described herein (for example, a monoclonalantibody molecule, and/or polyclonal antibody molecule, and/orbi-specific antibody molecule) comprises a detectable moiety and/or acytotoxic moiety.

By “detectable moiety”, we include one or more from the group comprisingof: an enzyme; a radioactive atom; a fluorescent moiety; achemiluminescent moiety; a bioluminescent moiety. The detectable moietyallows the antibody molecule to be visualized in vitro, and/or in vivo,and/or ex vivo.

By “cytotoxic moiety”, we include a radioactive moiety, and/or enzyme,for example wherein the enzyme is a caspase, and/or toxin, for examplewherein the toxin is a bacterial toxin or a venom; wherein the cytotoxicmoiety is capable of inducing cell lysis.

We further include that the antibody molecule may be in an isolated formand/or purified form, and/or may be PEGylated. PEGylation is a method bywhich polyethylene glycol polymers are added to a molecule such as anantibody molecule or derivative to modify its behavior, for example toextend its half-life by increasing its hydrodynamic size, preventingrenal clearance.

As discussed above, the CDRs of an antibody bind to the antibody target.The assignment of amino acids to each CDR described herein is inaccordance with the definitions according to Kabat E A et al. 1991, In“Sequences of Proteins of Immunological Interest” Fifth Edition, NIHPublication No. 91-3242, pp xv-xvii.

As the skilled person would be aware, other methods also exist forassigning amino acids to each CDR. For example, the InternationalImMunoGeneTics information system (IMGT®) (http://www.imgt.org/ andLefranc and Lefranc “The Immunoglobulin FactsBook” published by AcademicPress, 2001).

In some embodiments the antibody molecule that specifically binds TNFR2is a human antibody.

In some embodiments, the antibody molecule that specifically binds TNFR2is an antibody of human origin, i.e. an originally human antibody thathas been modified as described herein.

In some embodiments, the antibody molecule that specifically binds TNFR2is a humanized antibody, i.e. an originally non-human antibody that hasbeen modified to increase its similarity to a human antibody. Thehumanized antibodies may, for example, be of murine antibodies or lamaantibodies.

In some embodiments, the anti-TNFR2 antibody is a monoclonal antibody.

In some embodiments, the anti-TNFR2 antibody is a polyclonal antibody.

In some embodiments, the antibody molecule that specifically binds TNFR2comprises one of the VH-CDR1 sequences listed in Table 1 below.

In some embodiments, the antibody molecule that specifically binds TNFR2comprises one of the VH-CDR2 sequences listed in Table 1 below.

In some embodiments, the antibody molecule that specifically binds TNFR2comprises one of the VH-CDR3 sequences listed in Table 1 below.

In some embodiments, the antibody molecule that specifically binds TNFR2comprises one of the VL-CDR1 sequences listed in Table 1 below

In some embodiments, the antibody molecule that specifically binds TNFR2comprises one of the VL-CDR2 sequences listed in Table 1 below.

In some embodiments, the antibody molecule that specifically binds TNFR2comprises one of the VL-CDR3 sequences listed in Table 1 below.

In some embodiments the anti-TNFR2 antibody molecule is an antibodymolecule comprising the 6 CDRs having SEQ. ID. NOs: 1, 2, 3, 4, 5 and 6;or an antibody molecule comprising the 6 CDRs having SEQ. ID. NOs: 9,10, 11, 12, 13 and 14; or an antibody molecule comprising the 6 CDRshaving SEQ. ID. NOs: 17, 18, 19, 20, 21 and 22.

In some embodiments the anti-TNFR2 antibody molecule is an antibodymolecule comprising the 6 CDRs having SEQ. ID. NOs: 1, 2, 3, 4, 5 and 6.

In some embodiments, the anti-TNFR2 antibody molecule is an antibodymolecule selected from the group consisting of antibody moleculescomprising a VH selected from the group consisting of SEQ. ID. NOs: 7,15 and 23.

In some embodiments, the anti-TNFR2 antibody molecule is an antibodymolecule selected from the group consisting of antibody moleculescomprising a VL selected from the group consisting of SEQ. ID. NOs: 8,16 and 24.

In some embodiments the anti-TNFR2 antibody molecule is an antibodymolecule comprising a VH having SEQ. ID. NO: 7.

In some embodiments the anti-TNFR2 antibody molecule is an antibodymolecule comprising a VL having SEQ. ID. NO: 8.

In some embodiments the anti-TNFR2 antibody molecule comprises a VHhaving SEQ. ID. NO: 7 and a VH having SEQ. ID. NO: 8.

In some embodiments the anti-TNFR2 antibody molecule comprises a CHhaving SEQ. ID. NO: 217.

In some embodiments the anti-TNFR2 antibody molecule comprises a CLhaving SEQ. ID. NO: 218.

In some embodiments the anti-TNFR2 antibody molecule comprises a VHhaving SEQ. ID. NO: 7, a VH having SEQ. ID. NO: 8, a CH having SEQ. ID.NO: 217 and a CL having SEQ. ID. NO: 218.

TABLE 1Specific sequences of antagonistic TNFR2 blocking antibody molecules according to theinvention (in the VH and VL sequences, the CDR sequences are marked in bold text)Antibody SEQ. clone Region Sequence ID. NO: 001-H10 VH-CDR1FDDYGMSWVRQAPG 1 VH-CDR2 SVIYSGGSTYYADSVKGR 2 VH-CDR3 CARDRSSSWYRDGMDV 3VL-CDR1 CTGSSSNIGAGYDVH 4 VL-CDR2 GNSNRPS 5 VL-CDR3 CAAWDDSLSGWV 6 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVR- 7QAPGKGLEWVSVIYSGGSTYYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARDRSSS-WYRDGMDVWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNI- 8GAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAAWDDSLSGWVFGGGTKLTVLG 004-H02 VH-CDR1 FDDYGMSWVRQAPG 9 VH-CDR2STIYSGDNAYYGASVRGR 10 VH-CDR3 ARVYSSSWRKRAFDI 11 VL-CDR1 CSGTSSNIESNTVN12 VL-CDR2 SDNQRPS 13 VL-CDR3 CAAWDDSLSGWV 14 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVR- 15QAPGKGLEWVSTIYSGDNAYYGASVRGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCARVYSSSWRKRAFD- IWGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCSGTSSNI- 16 ESNTVNWYQQLPGTAPKLLIYSDNQRPSGVPDRFSG-SKSGTSASLAISGLR- SEDEADYYCAAWDDSLSGWVFGGGTKLTVLG 005-B08 VH-CDR1FSDYYMSWIRQAPG 17 VH-CDR2 AIISYDGGGKYFADPVKGR 18 VH-CDR3 ARYYGDGGFDP 19VL-CDR1 CTGSSSNIGAGYVVH 20 VL-CDR2 SNNQRPS 21 VL-CDR3 CAAWDDSLNGPV 22 VHEVQLLESGGGLVQPGGSLRLS- 23 CAASGFTFSDYYMSWIRQAPGKGLEWVAIISYDGGGKY-FADPVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARYYG- DGGFDPWGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCTGSSSNI- 24 GAGYVVHWYQQLPGTAPKLLIYSNNQRPSGVPDRFSG-SKSGTSASLAISGLR- SEDEADYYCAAWDDSLNGPVFGGGTKLTVLG

TABLE 2Specific sequences of TNFR2 blocking antibody molecules that are not antagonisticand that are used herein for comparison (in the VH and VL sequences, the CDRsequences are marked in bold text) 005-B02 VH-CDR1 FSDYYMSWIRQAPG 25VH-CDR2 ALIWYDGGNEYYADSVKGR 26 VH-CDR3 VRETGNYGMDV 27 VL-CDR1CTGSSSNIGAGYDVH 28 VL-CDR2 RNNQRPS 29 VL-CDR3 CATWDDRVNGPV 30 VHEVQLLESGGGLVQPGGSLRLS- 31 CAASGFTFSDYYMSWIRQAPGKGLEWVALIWYDGGNEY-YADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCVRETGNYGMDVWGQGT LVTVSS VLQSVLTQPPSASGTPGQRVTISCTGSSSNI- 32 GAGYDVHWYQQLPGTAPKLLIYRNNQRPSGVPDRFSG-SKSGTSASLAISGLRSEDEADYY- CATWDDRVNGPVFGGGTKLTVLG 001-E06 VH-CDR1FSSNYMSWVRQAPG 33 VH-CDR2 ALIWYDGSNKYYADSVKGR 34 VH-CDR3 AKDPLFDS 35VL-CDR1 CTGRSSNIGAGYDVH 36 VL-CDR2 DNNKRPS 37 VL-CDR3 CAAWDDSLNGPV 38 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSNYMSWVR- 39QAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCAKDPLFDSWGQGTLVT VSS VLQSVLTQPPSASGTPGQRVTISCTGRSSNI- 40 GAGYDVHWYQQLPGTAPKLLIYDNNKRPSGVPDRFSG-SKSGTSASLAISGLR- SEDEADYYCAAWDDSLNGPVFGGGTKLTVLG 001-G04 VH-CDR1FNTYSMNWVRQAPG 41 VH-CDR2 SVLYSDDDTHYADSVKGR 42 VH-CDR3ARDCGGDCHSGDDAFDI 43 VL-CDR1 CSGSSSNIGSNTVN 44 VL-CDR2 DNDKRPS 45VL-CDR3 CAAWHDSLNGWV 46 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYSMNWVR- 47QAPGKGLEWVSVLYSDDDTHYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARDCGG-DCHSGDDAFDIWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGSSS- 48NIGSNTVNWYQQLPGTAPKLLIYDNDKRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAAWHDSLNGWVLGGGTKLTVLG 001-G10 VH-CDR1 FSAYGMHWVRQAPG 49VH-CDR2 AVVSYDGREKHYADSVKGR 50 VH-CDR3 ARSDGGYDSDSGYY 51 VL-CDR1CSGSTSNIGSNFVY 52 VL-CDR2 DNNKRPS 53 VL-CDR3 CSSYAYSDNIL 54 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSAYGMHWVR- 55QAPGKGLEWVAVVSYDGREKHYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARSDG-GYDSDSGYYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGSTSNIGSNFVY- 56WYQQLPGTAPKLLIYDNNKRPSGVPDRFSG- SKSGTSASLAISGLRSEDEADYYCS-SYAYSDNILFGGGTKLTVLG 001-C08 VH-CDR1 FSNAWMSWVRQAPG 57 VH-CDR2SGISSSGSSAYYADSVKGR 58 VH-CDR3 ARHYYYHIAGYYYDTFDI 59 VL-CDR1CSGSSSNIGGNTVN 60 VL-CDR2 GNTNRPS 61 VL-CDR3 CAAWDDSLSGVV 62 VHEVQLLESGGGLVQPGGSLRLS- 63 CAASGFTFSNAWMSWVRQAPGKGLEWVSGISSSGS-SAYYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARHYYYHIAGYYYDTFD-IWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGSSS- 64NIGGNTVNWYQQLPGTAPKLLIYGNTNRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAAWDDSLSGVVFGGGTKLTVLG 001-H09 VH-CDR1 FSSYAMSWVRQAPG 65VH-CDR2 ATISYHGSDKDYADSVKGR 66 VH-CDR3 ARDANYHSSGYYYDVFDI 67 VL-CDR1CSGSSSNIGSNTVN 68 VL-CDR2 GNSNRPS 69 VL-CDR3 CAAWDDSLSTWV 70 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR- 71QAPGKGLEWVATISYHGSDKDYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARDANY-HSSGYYYDVFDIWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGSSS- 72NIGSNTVNWYQQLPGTAPKLLIYGNSNRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAAWDDSLSTWVFGGGTKLTVLG 005-F10 VH-CDR1 FSDYYMTWIRQAPG 73VH-CDR2 SGISGSGGYIHYADSVKGR 74 VH-CDR3 AREGLLPDAFD 75 VL-CDR1CSGSSSNIGNNYVS 76 VL-CDR2 RNNQRPS 77 VL-CDR3 CAAWDDSVSGWV 78 VHEVQLLESGGGLVQPGGSLRLS- 79 CAASGFTFSDYYMTWIRQAPGKGLEWVSGISGSGGYIHY-ADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCAREGLLPDAFD- IWGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCSGSSS- 80 NIGNNYVSWYQQLPGTAPKLLIYRNNQRPSGVPDRFSG-SKSGTSASLAISGLR- SEDEADYYCAAWDDSVSGWVFGGGTKLTVLG 001-B11 VH-CDR1FSSYSMNWVRQAPG 81 VH-CDR2 AVMSYDEYNTYYADSVKGR 82 VH-CDR3 AKGFYGDYPLWDY83 VL-CDR1 CSGGNSNIGTNTVD 84 VL-CDR2 SNNQRPS 85 VL-CDR3 CAAWDDSVNGPV 86VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVR- 87QAPGKGLEWVAVMSYDEYNTYYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCAKGFYGDY-PLWDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGGNSNIGTNTVDWYQQL 88PGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLR-SEDEADYYCAAWDDSVNGPVFGGGTKLTVLG 001-C07 VH-CDR1 FSSYEMNWVRQAPG 89VH-CDR2 STITGGGSIYDANSVQGR 90 VH-CDR3 ARDSTYHSSGYYYDVFDI 91 VL-CDR1CSGSSSNIGSNTVN 92 VL-CDR2 GNSNRPS 93 VL-CDR3 CAAWDDSLSGHWV 94 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVR- 95QAPGKGLEWVSTITGGGSIYDANSVQGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCARDSTYHSSGYYYDVFD IWGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCSGSSS- 96 NIGSNTVNWYQQLPGTAPKLLIYGNSNRPSGVPDRFSG-SKSGTSASLAISGLRSEDEADYYCAAWDDSLSGH- WVFGGGTKLTVLG 001-D01 VH-CDR1FSSYGMHWVRQAPG 97 VH-CDR2 SAVFGSGHGNTFYADAVKGR 98 VH-CDR3 AREQLWFGQDAFDI99 VL-CDR1 CSGSSSNIGSNTVN 100 VL-CDR2 GNSNRPS 101 VL-CDR3 CQSYDSSLSASV102 VH EVQLLESGGGLVQPGGPLRLSCAASGFTFSSYGMHWVR- 103QAPGKGLEWVSAVFGSGHGNTFYADAVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCAREQLWFGQDAFD- IWGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCSGSSS- 104 NIGSNTVNWYQQLPGTAPKLLIYGNSNRPSGVPDRFSG-SKSGTSASLAISGLR- SEDEADYYCQSYDSSLSASVFGGGTKLTVLG 003-F10 VH-CDR1FSDAWMTWVRQAPG 105 VH-CDR2 SDLSDSGGSTYYADSVKGR 106 VH-CDR3 GRLAAGGPVDY107 VL-CDR1 CTGSSSNIGAGYDVH 108 VL-CDR2 SNNQRPS 109 VL-CDR3 CSVWDDSLNSWV110 VH EVOLLESGGGLVQPGGSLRLSCAASGFTFSDAWMTWVR- 111QAPGKGLEWVSDLSDSGGSTYYADSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCGRLAAGGPVDYWGQGT LVTVSS VLQSVLTQPPSASGTPGQRVTISCTGSSSNI- 112GAGYDVHWYQQLPGTAPKLLIYSNNQRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCSVWDDSLNSWVFGGGTKLTVLG

In order to determine or demonstrate the features of the antibodymolecules of the present invention, they were compared to antibodymolecules that do not block TNFα ligand binding to TNFR2. Suchantibodies are shown in Table 3.

TABLE 3Specific sequences of non-blocking TNFR2 antibody molecules mentionedherein as reference antibodies(in the VH and VL sequences, the CDR sequences are marked in bold text)Antibody SEQ. clone Region Sequence ID. NO: 001-F02 VH-CDR1FSDYYMSWVRQAPG 113 VH-CDR2 ANINTDGSEKYYLDSVKGR 114 VH-CDR3 AREEYGAFDI115 VL-CDR1 CSGSSSNIGSNTVN 116 VL-CDR2 DNNKRPS 117 VL-CDR3 CQSFDRGLSGSIV118 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVR- 119QAPGKGLEWVANINTDGSEKYYLDSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCAREEYGAFD-IWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGSSS- 120NIGSNTVNWYQQLPGTAPKWYDNNKRPSGVPDRFSG- SKSGTSASLAISGLRSEDEADYYCQSFDR-GLSGSIVFGGGTKLTVLG 001-F06 VH-CDR1 FSSYAMHWVRQAPG 121 VH-CDR2SAISGGATTTYYADSVKGR 122 VH-CDR3 AKGGTGDPYYFDY 123 VL-CDR1CTGSSSNIGAGYDVH 124 VL-CDR2 RNNQRPS 125 VL-CDR3 CAARDDGLSGPV 126 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVR- 127QAPGKGLEWVSAISGGATTTYYADSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCAKGGTGDPYYFDYWGQ GTLVTVSS VLQSVLTQPPSASGTPGQRVTISCTGSSSNI- 128GAGYDVHWYQQLPGTAPKLLIYRNNQRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAARDDGLSGPVFGGGTKLTVLG 001-A09 VH-CDR1 FSSNYMSWVRQAPG 129VH-CDR2 SVISGSGGSTYYADSVKGR 130 VH-CDR3 ARDRGWFDP 131 VL-CDR1CSGSRSNIDNSYVS 132 VL-CDR2 RNNQRPS 133 VL-CDR3 CATwDDSLSGPV 134 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSNYMSWVR- 135QAPGKGLEWVSVISGSGGSTYYADSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCARDRGWFDPWGQGTLV TVSS VLQSVLTQPPSASGTPGQRVTISCSGSRSNIDNSYV- 136 SWYQQLPGTAPKLLIYRNNQRPSGVPDRFSG-SKSGTSASLAISGLRSEDEADYY- CATWDDSLSGPVFGGGTKLTVLG 001-B05 VH-CDR1FSNAWMSWVRQAPG 137 VH-CDR2 SSISSASGYIYYGDSVKGR 138 VH-CDR3 ARGTLYGDFDEF139 VL-CDR1 CSGSSSNIGNNAVN 140 VL-CDR2 GNTNRPS 141 VL-CDR3 CQSYDSSLSGYVV142 VH EVQLLESGGGLVQPGGSLRLS- 143CAASGFTFSNAWMSWVRQAPGKGLEWVSSISSASGYIY- YGDSVKGRFTISRD-NSKNTLYLQMNNLRAEDTAVYYCARGTLYGDFDEF- WGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCSGSSS- 144 NIGNNAVNWYQQLPGTAPKLLIYGNTNRPSGVPDRFSG-SKSGTSASLAISGLR- SEDEADYYCQSYDSSLSGYVVFGGGTKTVLG 001-B09 VH-CDR1FSRHAMNWVRQAPG 145 VH-CDR2 SSISTGSSYIDYADSVKGR 146 VH-CDR3 AREKGHYYYGMDV147 VL-CDR1 CTGSSSNIGAGYDVH 148 VL-CDR2 GNSYRPS 149 VL-CDR3CQSYDTSLSAYVV 150 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSRHAMNWVR- 151QAPGKGLEWVSSISTGSSYIDYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCAREKGHYYYG-MDVWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNI- 152GAGYDVHWYQQLPGTAPKLLIYGNSYRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCQSYDTSLSAYVVFGGGTKLTVLG 001-C03 VH-CDR1 FSNAWMSWVRQAPG 153VH-CDR2 SAISVSGINTYYADSVKGR 154 VH-CDR3 ARDTGSLGVDY 155 VL-CDR1CSGSSSNIGSNTVN 156 VL-CDR2 RNNQRPS 157 VL-CDR3 CQSYDSSLSISV 158 VHEVQLLESGGGLVQPGGSLRLS- 159 CAASGFTFSNAWMSWVRQAPGKGLEWVSAISVSGINTY-YADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARDTGSLGVDYWGQGT LVTVSS VLQSVLTQPPSASGTPGQRVTISCSGSSS- 160 NIGSNTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSG-SKSGTSASLAISGLR- SEDEADYYCQSYDSSLSISVFGGGTKLTVLG 001-C05 VH-CDR1FSSNEMSWIRQAPG 161 VH-CDR2 SVIYSGGSTYYADSVKGR 162 VH-CDR3 ARREGWLVPFDY163 VL-CDR1 CSGSSSNIGSNTVN 164 VL-CDR2 GNIIRPS 165 VL-CDR3 CQSFDTTLSGSIV166 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSNEMS- 167WIRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARREG-WLVPFDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGSSS- 168NIGSNTVNWYQQLPGTAPKLLIYGNIIRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCQSFDTTLSGSIVFGGGTKLTVLG 001-G05 VH-CDR1 FSSYAMSWVRQAPG 169VH-CDR2 SVISGSGGSTYYADAVKGR 170 VH-CDR3 TTDSGSGSYL 171 VL-CDR1CTGSSSNIGAGYDVH 172 VL-CDR2 SNNQRPS 173 VL-CDR3 CAAWDDSLNGPV 174 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR- 175QAPGKGLEWVSVISGSGGSTYYADAVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCTT-DSGSGSYLWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNI- 176GAGYDVHWYQQLPGTAPKLLIYSNNQRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAAWDDSLNGPVFGGGTKLTVLG 001-A10 VH-CDR1 FSDYYMTWIRQAPG 177VH-CDR2 SSISGGSTYYADSRKGR 178 VH-CDR3 AREPGYSYGFFDY 179 VL-CDR1CTGSSSNIGAGYDVH 180 VL-CDR2 SNNQRPS 181 VL-CDR3 CQSYDRSLSGSIV 182 VHEVQLLESGGGLVQPGGSLRLS- 183 CAASGFTFSDYYMTWIRQAPGKGLEWVSSISGGSTY-YADSRKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCAREPGY- SYGFFDYWGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCTGSSSNI- 184GAGYDVHWYQQLPGTAPKLLIYSNNQRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCQSYDRSLSGSIVFGGGTKLTVLG 001-C06 VH-CDR1 SSSYWMSWVRQAPG 185VH-CDR2 SAISGSGGSTYYADSVKGR 186 VH-CDR3 AREYSGYEFDF 187 VL-00R1CTGSSSNIGARSDVH 188 VL-CDR2 GNRNRPS 189 VL-CDR3 CQSFDRGLSGSIV 190 VHEVQLLESGGGLVQPGGSLRLSCAASGFTSSSYWMSWVR- 191QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCAR-EYSGYEFDFWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNI- 192GARSDVHWYQQLPGTAPKLLIYGNRNRPSGVPDRFSG- SKSGTSASLAISGLRSEDEADYYCQSFDR-GLSGSIVFGGGTKLTVLG 001-H03 VH-CDR1 FSSNYMSWVRQAPG 193 VH-CDR2SSISSSSSYIYYADSVKGR 194 VH-CDR3 ARDRGRTGTDY 195 VL-CDR1 CSGTTSNIGSYAVN196 VL-CDR2 GNINRPS 197 VL-CDR3 CQSYDSSLSASL 198 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSNYMSWVR- 199QAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARDRGRT-GTDYWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCSGTTSNIGSYAVNWYQQL 200PGTAPKLLIYGNINRPSGVPDRFSGSKSGTSASLAISGLR-SEDEADYYCQSYDSSLSASLFGGGTKLTVLG 004-E08 VH-CDR1 FSRYWMHWVRQVPG 201VH-CDR2 SGISDSGVVTYYADSVKGR 202 VH-CDR3 ARAQSVAFDI 203 VL-CDR1CSGSSSNIGAGHDVH 204 VL-CDR2 YDDLLPS 205 VL-CDR3 CAAWDDSLSGWV 206 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSRY- 207 WMHWVRQVPGKGLEWVSGISDSGVVTY-YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR- AQSVAFDIWGQGTLVTVSS VLQSVLTQPPSASGTPGQRVTISCSGSSSNIGAGH- 208DVHWYQQLPGTAPKLLIYYDDLLPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAAWDDSLSGWVFGGGTKLTVLG 005-A05 VH-CDR1 FSSYAMSWVRQAPG 209VH-CDR2 STIIGSGANTWYADSVKGR 210 VH-CDR3 ARHEGYYYYGMDV 211 VL-CDR1CTGSSSNIGAGYVVH 212 VL-CDR2 GNSNRPS 213 VL-CDR3 CAAWDDSLNGRV 214 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR- 215QAPGKGLEWVSTIIGSGANTWYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARHEGYYYYG-MDVWGQGTLVTVSS VL QSVLTQPPSASGTPGQRVTISCTGSSSNI- 216GAGYVVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSG- SKSGTSASLAISGLR-SEDEADYYCAAWDDSLNGRVFGGGTKLTVLG

The sequences in Tables 1, 2 and 3 above are all of human origin andderived from the n-CoDeR® library, as explained in detail in Example 1.

In some embodiments, the antibody molecules that specifically bind TNFR2described herein may also comprise one or both of the constant regions(CH and/or CL) listed in Table 4 below.

TABLE 4 SEQ. Region Sequence ID. NO: CH ASTKGPSVFPLAPSSKSTSGG- 217TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS-LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE-PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS-RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK-TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA- PIEK-TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI- AVEWESNGQPENNYKTTPPVLDSDGS-FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL- SPGK CLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW- 218KADSSPVKAGVETTTPSKQSNNKYAASSYLSLT- PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS CHAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT- 219WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVISSTWPSQSIT-CNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFIDPKIK-DVLMISLSPIVICVVVDVSEDDPDVOISWFVNNVEVHTAQ-TQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI-SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTN-NGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN- SYSCSVVHEGLHNHHTTKSFSRTPGK CHAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT- 220WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSIT-CNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIK-DVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ-TQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI-SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTN-NGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN- SYSCSVVHEGLHNHHTTKSFSRTPGK CLQPKSSPSVTLFPPSSEELETNKA- 221 TLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKY-MASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS

The first CH (SEQ. ID. NO: 217) and the first CL (SEQ. ID. NO: 218)sequences in Table 4 above are of human origin. The second CH (SEQ. ID.NO: 219) and the third CH (SEQ. ID. NO: 220) in Table 4 are both frommurine IgG2a, with that difference that the third CH sequence (SEQ. ID.NO: 220) contains an N297A mutation. The second CL sequence (SEQ. ID.NO: 221) is from murine lambda light chain constant region. These murinesequences are used in the examples for the surrogate antibodies.

In some embodiments, the antibody molecules bind human TNFR2 (hTNFR2).

In some embodiments, it is advantageous that the antibody molecule bindsboth to hTNFR2 and to cynomologus monkey TNFR2 (cmTNFR2 or cynoTNFR2).Cross-reactivity with TNFR2 expressed on cells in cynomolgus monkey,also called crab-eating macaque or Macaca fascicularis, may beadvantageous since this enables animal testing of the antibody moleculewithout having to use a surrogate antibody, with particular focus ontolerability.

In some embodiments, it is necessary to use a surrogate antibody to testan antibody molecule's functional activity in relevant in vivo models inmice. To ensure the comparability between the antibody molecule's effectin humans and the in vivo results for the surrogate antibody in mice, itis essential to select a functionally equivalent surrogate antibodyhaving the same in vitro characteristics as the human antibody molecule.

In some embodiments, the antibody molecule does not bind specifically toan epitope of TNFR2 comprising or consisting of the sequence KCSPG.

In some embodiments, the antibody molecule of the present invention orused according to the invention is an antibody molecule that is capableof competing with the specific antibodies provided herein, for examplecapable of competing with antibody molecules comprising a VH selectedfrom the group consisting of SEQ. ID. NOs: 7, 15 and 23; and/or a VLselected from the group consisting of SEQ. ID. NOs: 8, 16 and 24, forbinding to TNFR2.

By “capable of competing for” we mean that the competing antibody iscapable of inhibiting or otherwise interfering, at least in part, withthe binding of an antibody molecule as defined herein to the specifictarget TNFR2.

For example, such a competing antibody molecule may be capable ofinhibiting the binding of an antagonistic, blocking antibody moleculedescribed herein by at least about 10%; for example at least about 20%,or at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, about 100% and/or inhibiting the ability of theantibody described herein to prevent or reduce binding of TNFR2 to thespecific target ligand TNF-α by at least about 10%; for example at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or about 100%.

Competitive binding may be determined by methods well known to thoseskilled in the art, such as Enzyme-linked immunosorbent assay (ELISA).

ELISA assays can be used to evaluate epitope-modifying or blockingantibodies. Additional methods suitable for identifying competingantibodies are disclosed in Antibodies: A Laboratory Manual, Harlow &Lane, which is incorporated herein by reference (for example, see pages567 to 569, 574 to 576, 583 and 590 to 612,1988, CSHL, NY, ISBN0-87969-314-2).

In some embodiments, it is of interest to use not the antibody moleculeitself but a nucleotide sequence encoding such an antibody molecule. Thepresent invention thus encompasses nucleotide sequences encoding theabove antagonistic blocking TNFR-2 antibody molecules.

The above described antagonistic blocking antibody molecules andnucleotide sequences can be used in medicine, and then such an antibodymolecule and/or nucleotide sequence can be included in a pharmaceuticalcomposition, as discussed further below.

The above described antagonistic blocking antibody molecules, nucleotidesequences and/or pharmaceutical compositions can be used in thetreatment of cancer, as discussed further below.

The above described antagonistic blocking antibody molecules, nucleotidesequences and/or pharmaceutical compositions can be used in thetreatment of an infection caused by an intracellular pathogen, asdiscussed further below.

The above described antagonistic blocking antibody molecules and/ornucleotide sequences can be used in the manufacture of a pharmaceuticalcomposition for use in the treatment of cancer.

The above described antagonistic blocking antibody molecules and/ornucleotide sequences can be used in the manufacture of a pharmaceuticalcomposition for use in the treatment of an infection caused by anintracellular pathogen.

The above described antagonistic blocking antibody molecules and/orpharmaceutical compositions can be used in a method for treatment ofcancer in a patient, wherein a therapeutically effective amount of anantibody molecule or pharmaceutical composition is administered to thepatient.

The above described antagonistic blocking antibody molecules and/orpharmaceutical compositions can be used in a method for treatment of aninfection caused by an intracellular pathogen in a patient, wherein atherapeutically effective amount of an antibody molecule orpharmaceutical composition is administered to the patient.

In some embodiments relating to treatment of cancer, the cancer is asolid or leukemias cancer. A solid tumor is an abnormal mass of tissuethat usually does not contain cysts or liquid areas. Solid tumors may bebenign (not cancer), or malignant (cancer). Malignant solid tumors areherein denoted solid cancer. Different types of solid tumors are namedfor the type of cells that form them. Examples of solid tumors orcancers are sarcomas, carcinomas, and lymphomas.

More specific examples of solid cancers are lung cancer, breast cancer,colorectal cancer, prostate cancer, bladder cancer, ovarian cancer,endometrial cancer, kidney cancer, liver cancer, pancreatic cancer,thyroid cancer, brain cancer, central nervous system cancer, melanoma,neuroblastoma, Wilms tumor, rhabdomyosarcoma, retinoblastoma, head andneck cancer, gastric cancer, lymphoma and bone cancer.

More specific examples of leukemic cancers are acute lymphocyticleukemia, Chronic myeloproliferative disease, acute non-lymphocyticleukemia, B cell acute lymphocytic leukemia, chronic lymphocyticleukemia, T cell acute lymphocytic leukemia, non-Hodgkin lymphomas andchronic lymphoproliferative diseases.

In some embodiments relating to treatment of an infection caused by anintracellular pathogen, such as virus or bacteria. Specific examples ofintracellular pathogens are Legionella pneumophila, R. rickettsia,Mycobacterium tuberculosis, Listeria monocyotogenes, Salmonella spp,invasive Escherichia coli, Neisseria spp, Brucella spp, Shigella spp,Influenza virus, Herpes virus, Hepatitis virus, Coxsackievirus,Epstein-Barr virus or Rhinovirus.

In some embodiments relating to treatment of cancer, the above describedantagonistic blocking antibody molecules can be used in combination withan antibody molecule that specifically binds to a check-point inhibitor.Alternatively, the above discussed nucleotide sequences encoding ablocking TNFR2 antibody molecule can be used in combination withantibody molecule that specifically binds to a check-point inhibitor ora co-stimulatory agonistic antibody. Antibodies to check-pointinhibitors include antibodies targeting CTLA4, PD1, PD-L1, VISTA, TIGIT,CD200, CD200R, BTLA, LAG3, TIM3, B7-H3, B7-H4, B7-H7. Examples ofco-stimulatory agonistic antibodies are antibodies targeting OX40, 41BB,OX40L, 41BBL, GITR, ICOS, DR3, DR4, DR5, CD40, CD27, RANK, HVEM, LIGHTand B7-H6. Alternatively, the above discussed antagonistic blockingTNFR2 antibody molecules can be used in combination with a nucleotidesequence encoding an antibody molecule that specifically binds to acheck-point inhibitor or a costimulatory agonist. Alternatively, theabove discussed nucleotide sequences encoding a blocking TNFR2 antibodymolecule can be used in combination with a nucleotide sequence thatencodes an antibody molecule that specifically binds to a check-pointinhibitor or a costimulatory agonist. In some such embodiments, theantibody molecule that specifically binds to a check-point inhibitor isan anti-PD-1 antibody. PD-1 (or PD1) antibodies are thought to block theinhibitory signal mediated through PD-L1 primarily in CD8⁺ T cells; andthereby allowing an increased T cell mediated anti-tumor response. Anantagonistic TNFR2 antibody depleting Tregs would work through aseparate mechanism to increase the anti-tumor response. Hence thesetreatments could synergize with each other. The same is true for othercheck-point inhibitors and agonistic costimulatory antibodies.

Additionally, the above discussed antagonistic blocking TNFR2 antibodymolecules can be used in combination with other anti-cancer treatmentssuch as chemotherapy (e.g. but not limited to doxorubicin, paraplatin,cyclophosphamide, paditaxel, gemcitabine, 5-fluorouracil, docetaxel,vincristine, Mitoxantrone, mutamycin, epirubicin and methotrexate),small molecule tyrosin kinase or serine/threonine kinase inhibitors(e.g. but not limited to ibrutinib, imatinib, suntinib, regorafenib,sorafenib, dasatinib, eriotinib, vandetanib, midostaurin, vemurafenib,dabrafenib, palbociclib, ribociclib, Trametinib or alectinib),inhibitors targeting growth factor receptors (e.g. but not limited todrugs targeting EGFR/HER1/ErbB1, EGFR2/HER2/ErbB2, EGFR3/HER3/ErbB3,VEGFR, PDGFR HGFR, RET, insulin-like growth factor receptor IGFR, FGFR),anti-angiogenic agents (e.g. but not limited to Bevacizumab, Everolimus,Lenalidomide, Thalidomide, Zivaflibercept) or irradiation. Typically,the above mentioned anti-cancer drugs all cause cancer cell death whichwill lead to exposure of neo-antigens and inflammation. At a time whereneo-antigens are exposed, and there is an influx of inflammatory cellsin the tumor, there can occur synergistic effects of the anti-cancerdrug, and the addition of an antagonistic ligand blocking TNFR2antibody, which can deplete Tregs and thereby enhance the immune systemeven further.

It would be known to the person skilled in medicine, that medicines canbe modified with different additives, for example to change the rate inwhich the medicine is absorbed by the body; and can be modified indifferent forms, for example to allow for a particular administrationroute to the body.

Accordingly, we include that the antagonistic blocking antibodymolecules, nucleotide sequences, plasmids, viruses and/or cellsdescribed herein may be combined with a pharmaceutically acceptableexcipient, carrier, diluent, vehicle and/or adjuvant into apharmaceutical composition. In this context, the term pharmaceuticalcomposition can be used interchangeably with the terms pharmaceuticalpreparation, pharmaceutical formulation, therapeutic composition,therapeutic preparation, therapeutic formulation and therapeutic entity.

The pharmaceutical compositions described herein may comprise, or insome embodiments consist of, antibody molecules, nucleotide sequences,plasmids, viruses or cells.

The pharmaceutical compositions described herein may in some embodimentsconsist of or comprise plasmids comprising nucleotide sequences encodingthe above described antibody molecules or comprising the above describednucleotide sequences.

In some embodiments, the pharmaceutical compositions may comprisenucleotide sequences encoding parts of or a complete antibody moleculedescribed herein integrated in a cell or viral genome or in a viriome.The pharmaceutical composition may then comprise a cell or a virus as adelivery vehicle for an antibody of the invention (or a delivery vehiclefor a nucleotide sequence encoding an antibody of the invention). Forexample, in an embodiment, the virus may be in the form of a therapeuticoncolytic virus comprising nucleotide sequences encoding at least one ofthe antibody molecules described herein. In some embodiments, such anoncolytic virus comprises nucleotide sequences encoding a full-lengthhuman IgG antibody.

In some embodiments the invention relates to a virus comprising anucleotide sequence of the invention or a plasmid of the invention.Preferably, the virus is an oncolytic virus, such as a therapeuticoncolytic virus. Such viruses are known to those skilled in the arts ofmedicine and virology.

In some embodiments, such an oncolytic virus comprises nucleotidesequences encoding amino acid sequence having at least 80% identity witha sequence set out in table 1 above. In some embodiments, such anoncolytic virus comprises an amino acid sequence having at least 85%identity with a sequence set out in table 1 above. In some embodiments,such an oncolytic virus comprises an amino acid sequence having at least90% identity with a sequence set out in table 1 above, in someembodiments, such an oncolytic virus comprises an amino acid sequencehaving at least 95% identity with a sequence set out in table 1 above.

As an example, a nucleotide sequence encoding the antibody 001-H10 couldbe as presented in Table 5.

TABLE 5 Example of nucleotide sequences encoding the antibody 001-H10 -the parts of the sequences that are underlined in the tableencodes the VH and VL sequences, respectively, of 001-H10 SEQ. ID.Encoding Sequence NO: 001-H10 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGG- 222 VHTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAT-TCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAA- GCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG-TTATTTATAGCGGTGGTAGTACATATTAC-GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAA-GAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACAC-TGCCGTGTATTACTGTGCGAGAGATCGAAGCAGCAGCTGGTAC-CGCGATGGTATGGACGTCTGGGGCCAAGGTACACTGGTCAC-CGTGAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG-CACCCTCCTCCAAGAGCACCTCTGGGGG-CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC-CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG-CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG-CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGAC-CTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG- GACAAGAAAGTTGAGCCCAAATCTT-GTGACAAAACTCACACATGCCCACCGTGCCCAGCAC- CTGAACTCCTGGGGGGACCGTCAG-TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG-GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA-GACCCTGAGGICAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG-CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC-GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG-GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA-GCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG-CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG-GATGAGCTGACCAAGAACCAGGTCAGCCTGAC-CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG-GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC-GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAA-GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC-GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC-GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 001-H10CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGG- VLCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGG-CAGGTTATGATGTACACTGGTATCAGCAGCTCCCAGGAAC-GGCCCCCAAACTCCTCATCTATGGTAACAG- CAATCGGCCCTCAGGGGTCCCTGACCGAT-TCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAG-TGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGG-GATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGAACCAA-GCTGACGGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCAC-TCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACAC-TGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAG-TGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGA-GACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAG-CAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGC-TACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAG- TGGCCCCTACAGAATGTTCATGA

Some oncolytic viruses have capacity to host large enough DNA insertionsto accommodate integration of full-length human antibody sequences.Attenuated Vaccinia viruses and Herpes Simplex Viruses are examples oftherapeutic oncolytic viruses whose genome is sufficiently large topermit integration of full-length IgG antibody sequences (Chan et al.2014. ‘Oncolytic Poxviruses’, Annu Rev Virol, 1:119-41; Bommareddy. etal 2018. ‘Integrating oncolytic viruses in combination cancerimmunotherapy’, Nat Rev Immunol, 18: 498-513). Full-length IgGantibodies have successfully been integrated into oncolytic Vacciniavirus, resulting in expression and extracellular release (production) offull-length IgG antibodies upon infection of virus-susceptible hostcells e.g. cancer cells (Kleinpeter et al. 2016. ‘Vectorization in anoncolytic vaccinia virus of an antibody, a Fab and a scFv againstprogrammed cell death-1 (PD-1) allows their intratumoral delivery and animproved tumor-growth inhibition’, Oncoimmunology, 5: e1220467).Adenoviruses can also be engineered to encode full-length IgG antibodiesthat are functionally produced and secreted upon cellular infection(Marino et al. 2017. ‘Development of a versatile oncolytic virusplatform for local intra-tumoural expression of therapeutic transgenes’,PLoS One, 12: e0177810).

The invention also encompasses pharmaceutical compositions comprising avirus, such as an oncolytic virus, as discussed above, and apharmaceutically acceptable diluent, vehicle and/or an adjuvant.

The invention also comprises other therapeutic modalities, or “shapes”of drugs, such as antibody drug conjugates, fusion proteins etc., andpharmaceutical composition comprising such therapeutic modalities.

The antibody molecules, nucleotide sequences, plasmids, viruses, cellsand/or pharmaceutical compositions described herein may be suitable forparenteral administration including aqueous and/or non-aqueous sterileinjection solutions which may contain anti-oxidants, and/or buffers,and/or bacteriostats, and/or solutes which render the formulationisotonic with the blood of the intended recipient; and/or aqueous and/ornon-aqueous sterile suspensions which may include suspending agentsand/or thickening agents. The antibody molecules, nucleotide sequences,plasmids, cells and/or pharmaceutical compositions described herein maybe presented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (i.e.lyophilized) condition requiring only the addition of the sterile liquidcarrier, for example water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, and/or granules, and/or tablets of the kind previouslydescribed.

For parenteral administration to human patients, the daily dosage levelof the anti-TNFR2 antibody molecule will usually be from 1 mg/kgbodyweight of the patient to 20 mg/kg, or in some cases even up to 100mg/kg administered in single or divided doses. Lower doses may be usedin special circumstances, for example in combination with prolongedadministration. The physician in any event will determine the actualdosage which will be most suitable for any individual patient and itwill vary with the age, weight and response of the particular patient.The above dosages are exemplary of the average case. There can, ofcourse, be individual instances where higher or lower dosage ranges aremerited and such are within the scope of this invention.

Typically, a pharmaceutical composition (or medicament) described hereincomprising an antibody molecule will contain the anti-TNFR2 antibodymolecule at a concentration of between approximately 2 mg/ml and 150mg/ml or between approximately 2 mg/ml and 200 mg/ml.

Generally, in humans, oral or parenteral administration of the antibodymolecules, nucleotide sequences, plasmids, viruses, cells and/orpharmaceutical compositions described herein is the preferred route,being the most convenient. For veterinary use, the antibody molecules,nucleotide sequences, plasmids, viruses, cells and/or pharmaceuticalcompositions described herein are administered as a suitably acceptableformulation in accordance with normal veterinary practice and theveterinary surgeon will determine the dosing regimen and route ofadministration which will be most appropriate for a particular animal.Thus, the present invention provides a pharmaceutical formulationcomprising an amount of an antibody molecule, nucleotide sequence,plasmid, virus and/or cell of the invention effective to treat variousconditions (as described above and further below). Preferably, theantibody molecules, nucleotide sequences, plasmids, viruses, cellsand/or pharmaceutical compositions described herein is adapted fordelivery by a route selected from the group comprising: intravenous (IVor i.v.); intramuscular (IM or i.m.); subcutaneous (SC or s.c.) orintratumoral.

The present invention also includes antibody molecules, nucleotidesequences, plasmids, viruses, cells and/or pharmaceutical compositionsdescribed herein comprising pharmaceutically acceptable acid or baseaddition salts of the target binding molecules or parts of the presentinvention. The acids which are used to prepare the pharmaceuticallyacceptable acid addition salts of the aforementioned base compoundsuseful in this invention are those which form non-toxic acid additionsalts, i.e. salts containing pharmacologically acceptable anions, suchas the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate,bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, addcitrate, tartrate, bitartrate, sucdnate, maleate, fumarate, gluconate,saccharate, benzoate, methanesulphonate, ethanesulphonate,benzenesulphonate, p-toluenesulphonate and pamoate [i.e.1,1′-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.Pharmaceutically acceptable base addition salts may also be used toproduce pharmaceutically acceptable salt forms of the agents accordingto the present invention. The chemical bases that may be used asreagents to prepare pharmaceutically acceptable base salts of thepresent agents that are acidic in nature are those that form non-toxicbase salts with such compounds. Such non-toxic base salts include, butare not limited to those derived from such pharmacologically acceptablecations such as alkali metal cations (e.g. potassium and sodium) andalkaline earth metal cations (e.g. calcium and magnesium), ammonium orwater-soluble amine addition salts such asN-methylglucamine-(meglumine), and the lower alkanolammonium and otherbase salts of pharmaceutically acceptable organic amines, among others.The antibody molecules, nucleotide sequences, plasmids, viruses and/orcells described herein may be lyophilized for storage and reconstitutedin a suitable carrier prior to use. Any suitable lyophilization method(e.g. spray drying, cake drying) and/or reconstitution techniques can beemployed. It will be appreciated by those skilled in the art thatlyophilization and reconstitution can lead to varying degrees ofantibody activity loss (e.g. with conventional immunoglobulins, IgMantibodies tend to have greater activity loss than IgG antibodies) andthat use levels may have to be adjusted upward to compensate. In oneembodiment, the lyophilized (freeze dried) polypeptide binding moietyloses no more than about 20%, or no more than about 25%, or no more thanabout 30%, or no more than about 35%, or no more than about 40%, or nomore than about 45%, or no more than about 50% of its activity (prior tolyophilization) when re-hydrated.

The anti-TNFR2 antibody molecules, nucleotide sequences andpharmaceutical compositions described herein can be used use in thetreatment of cancer in a subject or patient. Herein, the terms subjectand patient are used interchangeably

“Patient” (or subject) as the term is used herein refers to an animal,including human, that has been diagnosed as having a specific disease.

In some embodiments, the patient (or subject) is an animal, includinghuman, that has been diagnosed as having cancer and/or that exhibitssymptoms of cancer.

In some embodiments, the patient (or subject) is an animal, includinghuman, that has been diagnosed as having an infection caused by anintracellular pathogen and/or that exhibits symptoms of an infectioncaused by an intracellular pathogen.

In some embodiments, the patient (or subject) is a patient having highTNFR2 expression in diseased tissue. In this context, high expressionmeans a higher level of TNFR2 expression compared to correspondinghealthy tissue. Normally the healthy tissue used for such a comparisonis a reference tissue (or standard reference) collected from healthytissue from one or several healthy individuals. The level of expressioncan be measured by standard techniques such as immunohistochemistry(IHC), fluorescence-activated cell sorting (FACS) or mRNA expressionmeasurements.

We include that the patient could be mammalian or non-mammalian.Preferably, the mammalian patient is a human, a horse, a cow, a sheep, apig, a camel, a dog or a cat. Most preferably, the mammalian patient isa human.

By exhibit symptoms of cancer, we include that the patient displays acancer symptom and/or a cancer diagnostic marker, and/or the cancersymptom and/or a cancer diagnostic marker can be measured, and/orassessed, and/or quantified.

It would be readily apparent to the person skilled in medicine what thecancer symptoms and cancer diagnostic markers would be and how tomeasure and/or assess and/or quantify whether there is a reduction orincrease in the severity of the cancer symptoms, or a reduction orincrease in the cancer diagnostic markers; as well as how those cancersymptoms and/or cancer diagnostic markers could be used to form aprognosis for the cancer.

Cancer treatments are often administered as a course of treatment, whichis to say that the therapeutic agent is administered over a period oftime. The length of time of the course of treatment will depend on anumber of factors, which could include the type of therapeutic agentbeing administered, the type of cancer being treated, the severity ofthe cancer being treated, and the age and health of the patient, amongstothers reasons.

By “during the treatment”, we include that the patient is currentlyreceiving a course of treatment, and/or receiving a therapeutic agent,and/or receiving a course of a therapeutic agent.

In some embodiments the cancer to be treated in accordance with thepresent invention is a solid tumor.

Each one of the above described cancers is well-known, and the symptomsand cancer diagnostic markers are well described, as are the therapeuticagents used to treat those cancers. Accordingly, the symptoms, cancerdiagnostic markers, and therapeutic agents used to treat the abovementioned cancer types would be known to those skilled in medicine.

Clinical definitions of the diagnosis, prognosis and progression of alarge number of cancers rely on certain classifications known asstaging. Those staging systems act to collate a number of differentcancer diagnostic markers and cancer symptoms to provide a summary ofthe diagnosis, and/or prognosis, and/or progression of the cancer. Itwould be known to the person skilled in oncology how to assess thediagnosis, and/or prognosis, and/or progression of the cancer using astaging system, and which cancer diagnostic markers and cancer symptomsshould be used to do so.

By “cancer staging”, we include the Rai staging, which includes stage 0,stage I, stage II, stage III and stage IV, and/or the Binet staging,which includes stage A, stage B and stage C, and/or the Ann Arbourstaging, which includes stage I, stage II, stage III and stage IV.

It is known that cancer can cause abnormalities in the morphology ofcells. These abnormalities often reproducibly occur in certain cancers,which means that examining these changes in morphology (otherwise knownas histological examination) can be used in the diagnosis or prognosisof cancer. Techniques for visualizing samples to examine the morphologyof cells, and preparing samples for visualization, are well known in theart; for example, light microscopy or confocal microscopy.

By “histological examination”, we include the presence of small, maturelymphocyte, and/or the presence of small, mature lymphocytes with anarrow border of cytoplasm, the presence of small, mature lymphocyteswith a dense nucleus lacking discernible nucleoli, and/or the presenceof small, mature lymphocytes with a narrow border of cytoplasm, and witha dense nucleus lacking discernible nucleoli, and/or the presence ofatypical cells, and/or cleaved cells, and/or prolymphocytes.

It is well known that cancer is a result of mutations in the DNA of thecell, which can lead to the cell avoiding cell death or uncontrollablyproliferating. Therefore, examining these mutations (also known ascytogenetic examination) can be a useful tool for assessing thediagnosis and/or prognosis of a cancer. An example of this is thedeletion of the chromosomal location 13q14.1 which is characteristic ofchronic lymphocytic leukemia. Techniques for examining mutations incells are well known in the art; for example, fluorescence in situhybridization (FISH).

By “cytogenetic examination”, we include the examination of the DNA in acell, and, in particular the chromosomes. Cytogenetic examination can beused to identify changes in DNA which may be associated with thepresence of a refractory cancer and/or relapsed cancer. Such mayinclude; deletions in the long arm of chromosome 13, and/or the deletionof chromosomal location 13q14.1, and/or trisomy of chromosome 12, and/ordeletions in the long arm of chromosome 12, and/or deletions in the longarm of chromosome 11, and/or the deletion of 11q, and/or deletions inthe long arm of chromosome 6, and/or the deletion of 6q, and/ordeletions in the short arm of chromosome 17, and/or the deletion of 17p,and/or the t(11:14) translocation, and/or the (q13:q32) translocation,and/or antigen gene receptor rearrangements, and/or BCL2 rearrangements,and/or BCL6 rearrangements, and/or t(14:18) translocations, and/ort(11:14) translocations, and/or (q13:q32) translocations, and/or (3:v)translocations, and/or (8:14) translocations, and/or (8:v)translocations, and/or t(11:14) and (q13:q32) translocations.

It is known that patients with cancer exhibit certain physical symptoms,which are often as a result of the burden of the cancer on the body.Those symptoms often reoccur in the same cancer, and so can becharacteristic of the diagnosis, and/or prognosis, and/or progression ofthe disease. A person skilled in medicine would understand whichphysical symptoms are associated with which cancers, and how assessingthose physical systems can correlate to the diagnosis, and/or prognosis,and/or progression of the disease. By “physical symptoms”, we includehepatomegaly, and/or splenomegaly.

BRIEF DESCRIPTION OF THE DRAWINGS

In the examples below, reference is made to the following figures:

FIG. 1 demonstrates that antibodies of the invention bind TNFR2. FIG. 1A-D: Human antibodies were shown by ELISA to bind to human TNFR2 proteinin a dose-dependent manner generating different EC50 values. FIG. 1 E:The murine antibodies 3-F10 and 5-A05 bind to mTNFR2 with a similaraffinity.

FIG. 2 shows binding of TNFR2 specific n-CoDeR® antibodies to in vitroactivated CD4⁺ T cells. Human blood derived CD4⁺ T cells (FIG. 2 A-D)and mouse splenic CD4⁺ T cells (FIG. 2 E) were activated with IL-2 andCD3/CD28 Dynabeads®. The affinity of TNFR2 specific n-CoDeR® antibodiesto activated cells were analyzed by FACS at concentrations ranging from0.002-267 nM (human) and 0.00003-133 nM (mouse). The curves show meanfluorescence intensity (MFI) after subtraction of isotype controlbackground (FIG. 2 A (complete and partial-blockers), FIG. 2 B(partial-blockers), Fig. C and D (non-blockers), FIG. 2 E (mousecomplete-blocker (3-F10) and non-blocker (5-A05)).

While human TNFR2 antibodies bind with different affinities to in vitroactivated CD4s (ranging EC50 values from 0.59 to 53 nM) the mouse TNFR2antibodies bind with similar affinity (EC50 values ranging from 0.072 to0.11 nM).

FIG. 3 shows that TNFR2 n-CoDeR® antibodies specifically bind to TNFR2.Human blood derived CD4⁺ T cells (FIG. 3 A) and mouse splenic CD4⁺ Tcells (FIG. 3 B) were activated 3 days with recombinant IL-2 andCD3/CD28 activation beads. In vitro activated cells were blocked with apolyclonal TNFR2 antibody (grey line) or left in PBS (black line) for 30min before stained with suboptimal concentration of the different TNFR2n-CoDeR® antibodies or isotype control (dashed line) for 15 min. Cellswere then washed and incubated with an APC conjugated secondary antibodyfor 30 min before analyzed by flow cytometry.

All antibodies could be blocked by the polyclonal TNFR2 antibody, henceshow that the TNFR2 n-CoDeR® antibodies (human and mouse) are specificto TNFR2.

FIG. 4 shows cross-reactivity of human TNFR2 specific n-CoDeR®antibodies to Cynomolgus. CD4⁺ T cells were isolated from cynomolgusblood and stimulated with PMA and lonomycin. After 2 days the cells werelabelled with 0.1, 1 or 10 μg/ml TNFR2 specific n-CoDeR® antibodies orisotype control followed by incubation with an APC conjugated secondarya-human antibody. Cells were analyzed by flow cytometry. The figureshows percentages of TNFR2⁺ T cells for the individual antibodies overthe isotype control. The results are the mean value and SD from 2-3individual experiments.

Most TNFR2 antibodies show cross-reactive binding to Cynomolgus cells.

FIG. 5 show that all herein described TNFR2 specific n-CoDeR® antibodiesbind other epitopes on the TNFR2 protein than the TNFR2 clone MR2-1.Human blood derived CD4⁺ T cells were stimulated with rhIL-2 andCD3/CD28 activation beads 2-3 days. Activated cells were either blockedby 40 μg/ml MR2-1 antibody (FIG. 5 A, black bars) or left with PBS (FIG.5 A, grey bars) 30 min, then TNFR2 specific n-CoDeR®antibodies/polyclonal TNFR2 (pTNFR2) were added and cells were incubated15 min. Percent bound TNFR2 n-CoDeR® antibodies were analyzed by FACSafter incubation with APC conjugated secondary antibodies. In FIG. 5 B,activated CD4⁺ T cells were blocked with 40 μg/ml TNFR2 specificn-CoDeR® antibodies/pTNFR2 (black bars) or left with PBS (grey bar) andthen incubated 15 min with PE conjugated MR2-1 antibody. Cells were thenanalyzed by FACS.

MR2-1 antibody did not interfere with the binding of the TNFR2 specificn-CoDeR® antibodies and the n-CoDeR® antibodies did not affect thebinding of MR2-1 to activated cells showing that the n-CoDeR® antibodiesall bind other domains of the TNFR2 protein than the MR2-1 antibody.

FIG. 6 shows ligand blocking activity of anti-human TNFR2 antibodies.Blocking ELISAs were performed with n-CoDeR® mAbs specific for hTNFR2 toevaluate ligand blocking characteristics. FIG. 6 A: All antibodies wereincubated at 10 μg/ml. Subsequently, all antibodies reducing the signalachieved with the isotype control by more than 50% (indicated by thedotted line) were dosed to further explore the ligand blockingpotential. FIG. 6 B shows complete blocking mAbs, FIGS. 6 C and D showpartially blocking mAbs and FIG. 6 E shows weak blocking mAbs. All othermAbs are considered non-blocking mAbs.

FIG. 7 shows ligand blocking activity of anti-mouseTNFR2 antibodies.Blocking ELISAs were performed with n-CoDeR® mAbs specific for mTNFR2 toevaluate ligand blocking characteristics. FIG. 7 A: All antibodies wereincubated at 10 μg/ml. Subsequently, all antibodies reducing the signalachieved with the isotype control by more than 50% (indicated by thedotted line) were dosed to further explore the ligand blockingpotential. FIG. 7 B shows complete blocking mAbs, FIGS. 7 C and D showpartially blocking mAbs and FIG. 7 E shows weak blocking mAbs. All othermAbs are considered non-blocking mAbs. Based on this, antibodies 3-F10and 5-A05 were chosen to represent complete blocking antibodies andnon-blocking antibodies respectively.

FIG. 8 Categorization of TNFR2 specific n-CoDeR® antibodies according totheir ability to agonize/antagonize TNFR2 signaling and ability to blockTNF-α binding to TNFR2. The ability of TNFR2 specific n-CoDeR®antibodies to enhance or reduce IFN-γ production was monitored usingIL-2 and IL-12 stimulated NK cells, and was plotted as a function ofantibodies ability to block TNF-α ligand binding to TNFR2 as describedabove. FIG. 8 A: Human blood derived NK cells were stimulated with 20ng/ml rhIL-2 and ng/ml rhIL-12 with the addition of 10 μg/ml TNFR2specific n-CoDeR® antibodies, isotype control or 100 ng/ml rhTNF-α for24 h. The amount of IFN-γ in the culture supernatants was measured usingMSD. The quantity of IFN-γ are normalized to isotype control (IFN-γvalues of isotype control=1 in figure) and shown in FIG. 8 A. Humanantibodies that had a higher EC50 value than 25 nM to in vitro activatedCD4⁺ T cells were not included in the analysis. FIG. 8 B: Human NK cellsalso produce TNF-α in these cultures (data shown mean TNF-α levels oftwo donors. The cell cultures supernatants were collected and theamounts of IFN-γ produced were analyzed using MSD. The results arenormalized against an isotype control. IFN-γ results are the mean valueof 3 donors in 2 independent experiments. The results reveal two extremegroups characterized by 1) antibodies with complete blocking andantagonist properties, and 2) antibodies with agonist non-blockingproperties, respectively. Agonistic non-blocking antibodies areagonistic and enhance the IFN-γ production from cytokine stimulated NKcells while blocking antibodies are antagonistic and inhibit IFN-γrelease. FIG. 8 C shows that the IFN-γ release is TNF-α dependent asneutralization of soluble TNF-α decreases the IFN-γ, while addingexogenous TNF-α increases IFN-γ. FIG. 8 D show that addition of ablocking anti-TNF-α antibody results in a dose-dependent neutralizationof soluble TNF-α. At a dose of 1 μg/ml, no soluble TNF-α can be detectedin the supernatant.

FIG. 9 shows that non-blocking agonistic but not blocking antagonisticTNFR2 specific n-CoDeR® antibodies enhance the proportion of CD25⁺ cellswithin the memory CD4⁺ T cells population. Human blood derived CD4⁺ Tcells (FIG. 9 A) and mouse splenic CD4⁺ T cells (FIG. 9 B) wereactivated with recombinant IL-2 and TNFR2 specific n-CoDeR® antibodies,isotype control or recombinant TNF-α. After 3 days of culture the cellswere stained for CD25 and CD45RO (human)/CD44 and CD62L (mouse) andanalyzed by flow cytometry. The results show the percentage of CD25expressing cells on the memory (CD45RO⁺ cells (human)/CD44⁺CD62L⁻(mouse)) population over the percentage of CD25⁺ cells recovered incultures with isotype control. The results are the mean value and SEM of7 donors (FIG. 9 A, human) and 3 mice (in 2 independent experiments,FIG. 9 B). In both human and mouse cultures non-blocking TNFR2antibodies induced the percentage of CD25⁺ memory cells, while blockingantibodies had no such effect on the memory population. For both humancultures (FIG. 9 A) and murine (FIG. 9 B), the addition of exogenousTNF-α increase the CD25⁺ memory T cell population*=p<0.05 as calculatedby one-way ANOVA.

FIG. 10 A shows that ligand blocking antagonistic antibodies have mostpronounced anti-tumor effect as mIgG2a, an isotype preferentiallyengaging activatory Fc receptors. Balb/c mice were injectedsubcutaneously with 1×10⁶ CT26 cells. After 8 days, at a mean tumor sizeof 3×3 mm mice were treated twice weekly with 10 mg/kg antibody i.p. asindicated in figures. Tumors were measured two times/week until theyreached a diameter of 15 mm, where after the mice were terminated. Theupper figure shows the tumor growth in isotype control treated mice,then the two figures below, in the left panel, show the antagonisticligand-blocker antibody (middle figure) and the agonistic non-ligandblocking antibody (lower figure) in FcγR defective Ig format. The middlepanel show the same antibodies in murine IgG2a format, engagingprimarily activatory FcγRs and the right panel the antibodies in murineIgG1 format engaging primarily the inhibitory FcγRIIb. For FIG. 10 Bsurviving mice were followed for 70 days. As seen in the figures, theblocking antagonistic antibody is most efficacious as tumor treatment inan IgG2a format engaging primarily activatory FcγRs and has no effect ina format with defective FcγR binding. On the other hand, thenon-blocking agonistic antibody is most efficacious as tumor treatmentin an IgG1 format preferentially engaging the inhibitory FcγR. Inaddition, the agonistic antibody has intrinsic, FcγR-independent,anti-tumor effects as seen using the N297A antibody format. ***=p<0.001compared to isotype control as calculated by Log-rank Mantel Cox test

FIG. 11 shows that ligand blocking antagonistic antibodies are effectiveas anti-tumor treatment in combination with anti-PD1. C57/BL6 mice wereinjected subcutaneously with 1×10⁶ MC38 cells. At a mean tumor size of3×3 mm mice were treated twice weekly with 10 mg/kg antibody i.p. asindicated in figures. The figure shows tumor growth curves of individualmice. FIG. 11 A: isotype control, FIG. 11 B: PD-1 targeting antibody,FIG. 11 C: 3-F10 antibody (surrogate antibody, ligand blocker,antagonist), FIG. 11 D: combination of 3-F10 and PD1. Tumors weremeasured two times/week until they reached a diameter of 15 mm, whereafter the mice were terminated. FIG. 11 E shows survival curves of thefour different treatment groups, **=p<0.01, ***=p<0.001 compared toisotype control as calculated by Log-rank Mantel Cox test.

FIG. 12 shows that blocking antagonistic antibodies are effective asanti-tumor treatment in combination with anti-PD-L1. C57/BL6 mice wereinjected subcutaneously with 1×10⁶ MC38 cells. At a mean tumor size of5×5 mm, mice were treated twice with isotype control antibody or 3F10(day 1 and 4), or four consecutive days with anti-PD-L1 followed by afifth injection two days later (in total five injections day 1, 2, 3, 4and 7), or a combination of both. All antibodies were administered at 10mg/kg i.p. Figure show mean tumor growth+/−SEM, n=10/group. *=p<0.05,***=p<0.001 as calculated using one-way ANOVA test.

FIG. 13: C57/BL6 mice were injected subcutaneously with 1×10⁶ B16.F10cells. After 3 days mice were treated twice weekly with 10 mg/kgantibody i.p. as indicated in figures. Tumors were measured twotimes/week until they reached a diameter of 15 mm, where after the micewere terminated. FIG. 13 A: isotype control, FIG. 13 B: 3-F10 antibody(surrogate antibody, ligand blocker, antagonist). FIG. 13 C showssurvival curves of the two different treatment groups. *=p<0.05 comparedto isotype control as calculated by Log-rank Mantel Cox test

FIG. 14 shows that ligand blocking antagonistic surrogate antibody 3F10alters immune cell composition in tumors. Mice were inoculated with CT26tumor cells as described and injected with antibodies as indicated oncethe tumors reached a size of approximately 7×7 mm. after 3 injections,at day 8 after treatment start, mice were sacrificed and tumorsharvested. Tumor single cell suspensions were analyzed for immune cellcontent by FACS. FIG. 14 A: Ligand blocking antagonistic surrogateantibody 3F10 causes Treg depletion and FIG. 14 B: CD8⁺ T cell influx orexpansion. This causes a shift in CD8⁺/Treg T cell ratio as depicted inFIG. 14 C. FIG. 14 D shows that not only T cells numbers but also thenumber of myeloid cells, here tumor associated macrophages (TAMs,defined as being CD11b⁺F4/80⁺MHCII⁺ but negative for both Ly6G and Ly6C)are very significantly reduced. The ligand non-blocking agonisticsurrogate antibody 5A05 also modulates TAM numbers but is stillsignificantly different from the ligand blocking antibody 3F10.

FIG. 15 shows that T cells in human tumors express levels of TNFR2similar to T cells retrieved from a PBMC reconstituted NOG mice.Briefly, NOG mice were injected i.v. with 15-20×10⁶ PBMC cells. After10-12 days, the spleens were removed from mice, single cell suspensionprepared and TNFR2 expression was assessed by FACS. Previously, TNFR2expression had been assessed on T cells retrieved from blood and tumorsamples from 3 or 9 cancer patients respectively. As shown in thefigure, the TNFR2 expression on Tregs and CD8⁺ T cells are verycomparable between the human T cells grown and activated in vivo in theNOG mice, and T cells from human tumors.

FIG. 16 shows that the ligand blocking antagonistic antibody 1-H10depletes Tregs in vivo in an FcγR dependent manner. NOG mice wereinjected i.v. with 15-20×10⁶ PBMC cells. After 10-12 days, the spleenswas removed from mice, single cell suspension prepared and then injectedi.p. into SCID mice. (10-15×10⁶/mice). After 1 h, mice were treated with10 mg/kg antibody i.p. and 24 hours after antibody injection, i.p. fluidwas collected from mice and the cells in the fluid were analyzed usingFACS. FIG. 16 A shows the mean percentage of stained Tregs (defined asCD45⁺CD3⁺CD4⁺CD25⁺CD127^(low/neg)) out of the human CD45⁺ population andshow that Tregs are significantly depleted by the blocking antibody1-H10. FIG. 16 B shows the mean percentage of CD8⁺ T out of the humanCD45⁺ and shows that 1-H10 significantly increases the CD8⁺ T cellpopulation. FIG. 16 C shows that the ratio of CD8⁺ T cells against theTregs is significantly increased by 1-H10. Data in FIG. 16 A-C ispresented as mean of four different experiments where each dotrepresents one mouse. Yervoy and a commercially available anti-CD25antibody were used as positive controls. All data is normalized toisotype control so that in FIGS. 16 A and B isotype control is set to100% and in FIG. 16 C to 1. FIG. 16 D shows a separate experiment whereFcγR binding defective 1-H10 IgG1N297Q antibody was used (designated1-H10NQ in the figure) to assess the dependency of FcγR binding for Tregdepletion. As shown in the figure, the depletion is most efficacious inthe wild type IgG1 format (1-H10) compared to the Fc defective(1-H10NQ).

FIG. 17 shows that antagonistic ligand non-blocking TNFR2 antibodies donot induce cytokine release in vitro. IFN-γ release induced by variousTNFR2 specific antibodies was measured in three different in vitrosystems. As positive controls, and anti-CD3 antibody=OKT3, an anti-CD52antibody=Alemtuzumab and an anti-CD28 antibody were used. Isotypecontrol was used as a negative control. Each dot represents PBMC fromone human donor. FIG. 17 A shows results from high density cell cultureswere PBMCs were cultured at 1×10⁷ cells/ml. After 48 h, 10 μg/mlantibody was added and incubated for 24 h. As seen in the figure, bothAlemtuzumab and OKT3 induced significant IFN-γ release but not any ofthe TNFR2 specific antibodies. FIG. 17 B shows Solid Phase in vitrocultures performed by coating wells of a 96-well plate with antibodiesbefore adding PBMCs. Again, both Alemtuzumab and OKT3 inducedsignificant IFN-γ release along with some of the TNFR2 specificantibodies. However, the complete blocking antibody 1-H10 did not inducecytokine release above isotype control antibody. FIG. 17 C showsstimulation of whole blood with antibody and here Alemtuzumab inducedsignificant IFN-γ release but not any of the TNFR2 specific antibodies.

FIG. 18 shows that antagonistic ligand non-blocking TNFR2 antibodies donot induce cytokine release in vivo. NOG mice were injected i.v. with25-×10⁶ PBMC cells. After 14 days, when the blood of the mice was shownto consist of approximately 40% human T-cells, mice were treated with 10μg antibody. Body temperature was measured 1 h post injection (FIG. 18A). The experiment was terminated 5 h post injection and blood wasanalyzed for IFN-γ (FIG. 18 B) or TNF-α (FIG. 18 C) content.****=p<0.0001 and **=p<0.01 as calculated with one-way ANOVA.

FIG. 19 shows binding to TNFR2 variants lacking individual domains.Antibody binding to TNFR2 variants expressed on HEK cells were tested ina flow cytometry approach. Lack of domain 1 and 2 does not significantlyaffect binding (FIGS. 19 A and B), while 3 and partially 4 completelyabrogates interaction between the antibody and TNFR2 (FIGS. 19 C and D).Similarly lack of domain 1+3 prevents binding of all antibodies (except1F06) completely (FIG. 19 E), while the lack of domain 2+4 abrogatebinding completely for the agonistic antibodies (1F02, 1F06, 4E08) andsignificantly reduces binding also for the antagonists (1H10, 4H02,5B08) (FIG. 19 F). Dark grey indicates positive control and whitenegative control antibody.

FIG. 20 shows a comparison of amino acid sequence of human (H-D3) andmouse (M-D3) domain 3 of TNFR2. Similar amino acids are marked in white,while differences are marked in grey. The five sequences below representthe 5 different constructs the antibodies are tested against. Exchangesof human-to-mouse sequence are underlined, while the non-marked sequenceis completely human. Domains 1, 2 and 4 are human and do not contain anysubstitutions or mutations.

FIG. 21 shows binding to wild type human and mouse TNFR2 (left panels).Mutated hTNFR2 constructs (m1, m2, m3 and m4) were used to narrow downthe binding site for different anti hTNFR2 antibodies. Flow cytometryanalysis revealed that mutations in aa 119-132 do not affect antibodybinding, while mutations in aa 151-160 completely abrogate binding ofall antibodies. Mutations in 134-144 disrupt binding for blocking andantagonistic antibodies only but does not affect the agonisticantibodies significantly. Dark grey bars indicate positive control andwhite negative control antibody. Dashed line is the level of thenegative control antibody.

EXAMPLES

Specific, non-limiting examples which embody certain aspects of theinvention will now be described.

In many of the examples, in particular the in vivo examples, theantibody 3-F10 has been used. This is a mouse antibody, which is asurrogate antibody to the human antibodies disclosed herein. It has beenselected based on its ability to bind murine TNFR2, its blocking ofmurine TNF-α ligand binding to TNFR2, and based on its antagonisticactivity in a murine T cell activation assay as described in example 4.In some examples, the 3-F10 antibody was tested and compared indifferent antibody formats associated with strong and preferentialbinding to activatory over inhibitory Fc gamma receptors (mIgG2a),strong and preferential binding to mouse inhibitory FcγR (mIgG1) ordefective binding to mouse FcγR (mIgG2a N297A).

In some examples, the antibody 5-A05, has been used. This is a mousesurrogate antibody for the human anti-TNFR2, non-blocking, agonisticantibodies included herein for reference and for comparative reasons.5-A05 was selected as surrogate based on its ability to bind murineTNFR2, lack of blocking effects on murine TNF-α ligand binding to TNFR2,and on its agonistic activity in a murine T cell activation assay asdescribed in example 4. In some examples, the 5-A05 antibody was testedand compared in different antibody formats associated with strong andpreferential binding to activating over inhibitory Fc gamma receptors(mIgG2a), strong and preferential binding to mouse inhibitory FcγR overactivatory FcγR (mIgG1) or defective binding to mouse Fcγ (N297A).

In some of the examples and figures a slightly different naming of theantibody clones is used, for example, clone 001-H10 is sometimesshortened to 1-H10 or 1H10, 005-B08 is sometimes shortened to 5-B08 or5B08 etc.

Example 1—Generation of TNFR2 Specific Antibodies

(See also FIG. 1 and the above description of this figure.)Isolation of scFv Antibody Fragments

The n-CoDeR® scFv library (BioInvent, Söderlind E, et al Nat Biotechnol.2000; 18(8):852-6) was used to isolate scFv antibody fragmentsrecognizing human or mouse TNFR2.

The phage library was used in three consecutive pannings againstrecombinant human or mouse protein (Sino Biological). After phageincubation, the cells were washed to remove unbound phages. Bindingphages were eluted with trypsin and amplified in E. coli. The resultingphage stock was converted to scFv format. E. coli was transformed withscFv bearing plasmids and individual scFv clones were expressed.

Identification of Unique TNFR2 Binding scFv

Converted scFv from the third panning were assayed using a homogeneousFMAT analysis (Applied Biosystems, Carlsbad, Calif., USA) for binding to293 FT cells transfected to express human or mouse TNFR2 or anon-related protein.

Briefly, transfected cells were added to clear-bottom plates, togetherwith the scFv-containing supernatant from expression plates (diluted1:7), mouse anti-His Tag antibody (0.4 μg/ml; R&D Systems) and anAPC-conjugated goat anti-mouse antibody (0.2 μg/ml; cat. no.115-136-146, Jackson Immunoresearch). FMAT plates were incubated at roomtemperature for 9 h prior to reading. Bacterial clones binding TNFR2transfected cells but non cells transfected with a non-related proteinwere classified as actives and cherry picked into 96-well plate.

IgG Binding to TNFR2 in ELISA

96-well plates (Lumitrac 600 LIA plate, Greiner) were coated overnightat 4° C. with recombinant human or mouse TNFR2-Fc protein (SinoBiological) at 1 pmol/well. After washing, titrated doses of anti-TNFR2mAbs from 20 μg/ml to 0.1 ng/ml (133 nM to 1 pM) were allowed to bindfor 1 hour. Plates were then washed again, and bound antibodies weredetected with an anti-human-F(ab)-HRP secondary antibody (JacksonImmunoResearch) diluted in 50 ng/ml. Super Signal ELISA Pico (ThermoScientific) was used as substrate and the plates were analyzed usingTecan Ultra Microplate reader.

The data, which are shown in Table 6 and in FIG. 1 A-D, show that thehuman anti-TNFR2 antibodies all bind to human TNFR2 protein. The EC50values are ranging from 0.082 nM for 1-C08 to 4.4 nM for 1-A09.

In addition, the mouse antibody surrogate clones 3-F10 and 5-A05 alsobind to mTNFR2 protein. These two clones bind with a very similaraffinity (Table 6 and FIG. 1E).

TABLE 6 EC50 values of antibodies binding to TNFR2 protein (humanprotein except for clone 3F10 and 5A05) Clone EC₅₀ (nM) 1-C08 0.0821-E06 0.20 1-G10 0.29 1-H10 0.29 4-H02 0.20 5-B02 0.15 5-B08 0.17 1-G041.7 1-H09 0.30 1-D01 0.37 5-F10 0.22 1-B11 0.25 1-C07 0.26 1-B05 0.231-F02 0.31 1-F06 0.15 4-E08 0.38 1-G05 0.54 1-A09 4.4 1-B09 0.18 1-C030.75 1-C05 0.38 3-F10 (mouse) 0.97 5-A05 (mouse) 1.4

Example 2—Specificity of Antibodies

(See also FIGS. 2-5 and the above description of these figures.)Isolation of CD4⁺ T cells

PBMCs from human buffy coats and Cynomolgus macaques (M. fascicularis)whole blood were isolated using Ficoll-Paque PLUS (GE Healthcare)gradients. CD4⁺ T cells were isolated from PBMCs by magnetic cellsorting using CD4⁺ T cell isolation Kit (human) or CD4 MicroBeads,non-human primate (Cynomolgus) both from Miltenyi. Mouse CD4⁺ T cellswere isolated from spleen using CD4⁺ T cell isolation kit (mouse) fromMiltenyi.

Titration of TNFR2 Specific n-CoDeR® Antibodies

The ability and affinity of TNFR2 n-CoDeR® antibodies to bind TNFR2expressed on cells were obtained using in vitro activated CD4⁺ T cells.Human CD4⁺ T cells were stimulated with 50 ng/ml rhIL-2 (R&D systems)and Dynabeads® T-Activator CD3/CD28 for T-Cell Expansion and Activation(Gibco) 2-3 days at 37° C. In vitro activated cells were labelled withincreasing amount of n-CoDeR® antibodies specific for TNFR2 or isotypecontrol, ranging from 0.002-267 nM. Cells were then incubated with anAPC conjugated a-human IgG secondary ab (Jackson) followed by analysisby flow cytometry (FACSVerse, BD). The resulting titration curves areshown in FIG. 2 A-D. Mouse CD4⁺ T cells were stimulated with 135 U/mlrmIL-2 (R&D systems) and Dynabeads® T-Activator CD3/CD28 for T-CellExpansion and Activation (Gibco) 2-3 days at 37° C. In vitro activatedcells were labelled with increasing amount of n-CoDeR® antibodiesspecific for TNFR2 or isotype control, ranging from 0.00003-133 nM.Cells were then incubated with an APC conjugated a-mouse IgG secondaryab (Jackson) followed by analysis by flow cytometry (FACSVerse, BD). Thetitration curves are shown in FIG. 2E. The EC50 values for the titrationcurves were calculated in Microsoft Excel and are shown in Table 7. Forthe human antibodies the EC50 values differed from 0.6 nM (4-H02) to52.7 nM (1-C03). The mouse antibodies bound to in vitro activated cellswith similar affinity (0.072 nM (3-F10) and 0.11 nM (5-A05)).

Specificity of TNFR2 n-CoDeR® Antibodies

The specificity of TNFR2 antibodies to TNFR2 were obtained in FACSblocking experiments with a commercial polyclonal TNFR2 antibody (R&Dsystems). CD4⁺ T cells (mouse and human) stimulated 2-3 days with 50ng/ml rhIL-2 (R&D systems) (human)/135 U/ml rm IL-2 (R&D systems)(mouse) and Dynabeads® T-Activator CD3/CD28 for T-Cell Expansion andActivation (Gibco) were blocked with 40 μg/ml polyclonal TNFR2 antibody(R&D systems) for 30 min, immediately followed by 15 min incubation withTNFR2 n-CoDeR® antibodies or isotype control. The concentration ofn-CoDeR® antibodies used was based on the titration curves for theindividual TNFR2 n-CoDeR® antibodies and a suboptimal concentration foreach antibody was chosen. Cells were then washed and incubated 30 minwith an APC conjugated secondary antibody (Jackson). Cells were analyzedby flow cytometry (FACSVerse, BD). All binding of TNFR2 specificn-CoDeR® antibodies (both human and mouse) could be blocked by apolyclonal TNFR2 antibody as shown in FIG. 3. These results verifyingthat TNFR2 n-CoDeR® antibodies specifically bind TNFR2 on in vitroactivated CD4⁺ T cells.

Epitope Mapping of TNFR2 Specific n-CoDeR® Antibodies Against the TNFR2Antibody Clone MR2-1

The TNFR2 antibody clone MR2-1 (Invitrogen) binds a specific domain ofthe TNFR2 protein. If the TNFR2 specific n-CoDeR® antibodies bound tothe same domain as MR2-1 was tested by FACS blocking experiments.

Human CD4⁺ T cells were stimulated 2-3 days with 50 ng/ml rhIL-2 (R&Dsystems) and Dynabeads® T-Activator CD3/CD28 for T-Cell Expansion andActivation (Gibco). Activated cells were blocked with 40 μg/ml MR2-1(black bars in FIG. 5 A), or PBS (grey bars in FIG. 5). After 30 minincubation, cells were immediately stained for TNFR2 specific n-CoDeR®antibody or polyclonal TNFR2 (pTNFR2) for 15 min. After incubation withan APC conjugated secondary anti-human IgG reagent (Jackson), cells wereanalyzed by flow cytometry (FACSVerse, BD). In FIG. 5 B, activated CD4⁺T cells were blocked with 40 μg/ml TNFR2 specific n-CoDeR® antibodies orpTNFR2 (black bars) or left with PBS (grey bar) and then incubated 15min with PE conjugated MR2-1 antibody. Again, cells were then analyzedby FACS. Since the percentage of MR2-1+ cells were the same for n-CoDeR®blocked as non-blocked cells (FIG. 5 B) and the binding of n-CoDeR®antibodies were the same with or without MR2-1 block (FIG. 5 A) thesedata show that the n-CoDeR® antibodies bind other epitopes of the TNFR2protein than the MR2-1 antibody.

Binding Off TNFR2 n-CoDeR® Antibodies to Cynomolgus

To validate the cross-reactivity of TNFR2 antibodies to Cynomolgus,Cynomolgus CD4⁺ T cells were stimulated 2 days with 50 ng/ml PMA (Sigma)and 100 ng/ml lonomycin (Sigma) to cause upregulation of TNFR2. Cellswere incubated with TNFR2 specific n-CoDeR® antibodies at 3 differentconcentrations (0.1, 1 and 10 μg/ml) and then incubated with an APCconjugated secondary a-human IgG reagent (Jackson). Cells were analyzedby flow cytometry (FACSVerse, BD) and the result show that most of thehuman TNFR2 specific n-CoDeR® antibodies could bind Cynomolgus TNFR2,the results for the individual antibodies are presented in FIG. 4.

In summary, the data in example 2 show that the human antibodiesspecifically bind to TNFR2 endogenously expressed on human immune cells.Furthermore, the data show that this binding can be blocked by adding apolyclonal commercially available antibody against TNFR2, whichindicates very high specificity for TNFR2. The same is true for thesurrogate clones 3F10 and 5A05 regarding murine cells expressing murineTNFR2. Also, the binding of the human clones is unaffected by MR2-1antibodies showing a different epitope specificity compared to MR2-1.

TABLE 7 EC₅₀ values calculated on the titration of TNFR2 specificantibodies to in vitro activated CD4⁺ T cells. Clone EC₅₀ (nM) 1-C08 2.61-E06 4.1 1-G10 3.3 1-H10 1.1 4-H02 0.59 5-B02 0.80 5-B08 1.2 1-G04 181-H09 16 1-D01 3.9 5-F10 32 1-B11 27 1-C07 36 1-B05 1.5 1-F02 0.79 1-F062.5 4-E08 2.3 1-G05 0.66 1-A09 48 1-B09 29 1-C03 53 1-C05 12 3-F10(mouse) 0.072 5-A05 (mouse 0.11

Example 3—Test of Ligand Blocking Characteristics

(See also FIGS. 6-7 and the above description of these figures.)

ELISA Method

96-well plates were coated with hTNFR2 (Sinobioologicals Cat. No.10414-H08H) or mTNFR2 (Sinobioologicals Cat. No. 50128 M08H) at 2.5pmol/well in ELISA coating buffer (0.1 M sodium carbonate pH 9.5) andincubated overnight at 4° C. After washing in ELISA wash buffer (PBSwith 0.05% Tween20), the plates were incubated under slow agitation for1 h in room temperature with n-CoDeR® mAbs at 10 μg/ml (one-dose ELISA)or 33 nM and subsequent 1:2 dilutions (titration ELISA) in block buffercontaining 0.45% fish gelatin. Subsequently, recombinant hTNF-α-bio (R&DCat. No. BT210) or mTNF-α (Gibco Cat. No. PMC3014) were added at a finalconcentration of 5 nM and 2 nM respectively and allowed to incubate foranother 15 min. Thereafter, plates were washed. For the human ELISAs,Streptavidin-HRP (Jackson Cat. No. 016-030-084) diluted 1:2000 in blockbuffer were added and again incubated for 1 h at room temperaturefollowed by washes first in ELISA buffer and then in Tris buffer (pH9.8). The substrate (Super Signal ELISA Pico from Thermo Scientific Cat.No. 37069) were thereafter diluted according to the manufacturersinstruction, added to the wells and incubated in darkness for 10 minbefore reading in a Tecan Ultra. For the mouse ELISAs, rabbitanti-mTNF-α (Sinobiologicals Cat. No. 50349-RP02) diluted to 1 μg/ml inwas added and allowed to incubate for 1 h in room temperature. Afterwashing, anti-Rabbit-HRP diluted 1:10 000 in block buffer was added andagain incubated for 1 h at room temperature. Substrate adding andreading were performed as above.

The data is presented in Tables 8 and 9 below, and in FIGS. 6 and 7.

TABLE 8 EC50 values of ligand blocking human antibodies: Antibodies weretitrated, and EC 50 values were calculated. Clone EC50 (nM) Block001-H10 0.9 Complete 004-H02 0.4 Complete 005-B08 0.3 Complete 005-B020.2 Complete 001-E06 0.3 Partial 001-G10 1.6 Partial 001-C08 1.1 Partial001-H09 1.4 Partial 005-F10 0.03 Partial 001-G04 3.2 Partial 001-B11 1.0Weak 001-C07 0.8 Weak 001-D01 1.4 Weak

TABLE 9 EC50 values of ligand blocking murine antibodies: Antibodieswere titrated, and EC 50 values were calculated. Clone EC50 (nM) Block3-F10 1.9 Complete 4-C01 2.7 Complete 4-A06 2.0 Partial 4-A07 >500Partial 4-F06 6.2 Partial 5-C09 8.6 Partial 2-D09 4.4 Partial 4-B12 >500Partial 3-G06 13 Partial 2-H01 25 Weak 4-C02 >500 Weak 4-G09 2.6 Weak4-C03 8.3 Weak

Blocking Definitions

-   -   Complete blockers are defined as reducing the TNF-α binding with        more than 98%    -   Partial blockers are defined as reducing the TNF-α binding with        60-98%    -   Weak blockers are defined as reducing the TNF-α binding with        less than 60%    -   Non-blocking antibodies are defined as not reaching more than        50% block in high-dose, one-point ELISA as shown in FIGS. 6A and        7A

The data shown in this example show that various antibodies have beengenerated ranging from antibodies which completely inhibit the ligandTNF-α from binding to antibodies that do not inhibit ligand blocking atall. This is true for both human antibodies and murine surrogates.

Example 4—In Vitro Functionality of Antibodies

(See also FIGS. 8-9 and the above description of these figures.)

TNFR2 Antibodies Ability to Regulate Cytokine Stimulated NK Cells IFN-γProduction

The agonistic/antagonistic characteristics of TNFR2 specific antibodieswere evaluated using a NK cell assay described by Almishri et al. (TNFαAugments Cytokine-Induced NK Cell IFNγ Production through TNFR2.Almishri W. et al. J Innate Immun. 2016; 8:617-629).

In brief, human NK cells were isolated from human PBMCs by MACS using“NK isolation kit” (Miltenyi). 100 μl NK cells (1×10⁶ cells/ml) werecultured with 20 ng/ml rhIL-2 (R&D systems) and 20 ng/ml rhIL-12 (R&Dsystems) together with 10 μg/ml TNFR2 specific antibodies, 10 μg/mlisotype control or 100 ng/ml TNF-α (R&D systems) in U-bottom plates(Corning® 96 Well TC-Treated Microplates, Sigma-Aldrich). Supernatantswere collected after 24 h and the amount of IFN-γ produced was assessedby MSD.

As control, anti-TNF-α antibody neutralizing TNF-α (Cat No. AF-210-NA,R&D systems) was included. As seen in FIG. 8 D, a dose of 1 μg/mlcompletely neutralized soluble TNF-α and this dose also lowered theIFN-γ release.

Human non-blocking TNFR2 antibodies distinctly enhanced the IL-2 andIL-12 stimulated NK cells IFN-γ production (2-3 times more IFN-γ thanisotype control) while antagonistic antibodies (here shown by completeblockers) showed antagonistic effects on NK cells and decreased IFN-γproduction (FIG. 8 A).

This test was considered non-representative to perform with the mousesurrogate antibodies due to lack of endogenously produced TNF-α in themurine cultures, as well as expression of inhibitory FcγR on murine NKcells while only activating FcγR was expressed on the human counterpart.Instead, the memory T cell activation assay (induction of CD25) asdescribed below was used to address the agonist or antagonisticproperties of the murine surrogate antibodies.

Induction of CD25 Expressing Memory CD4⁺ T cells by TNFR2 antibodies

To further understand the agonistic/antagonistic characteristics of theTNFR2 antibodies, their ability of enhancing the proportion of CD25expressing memory CD4⁺ T cells were evaluated.

Briefly, human CD4⁺ T cells were isolated from PBMCs by MACS using the“CD4⁺ T cell isolation kit” from Miltenyi. CD4s were cultured with 10ng/ml rhIL-2 (R&D systems) and 10 μg/ml TNFR2 specific antibody orindicated amount of rhTNF-α (R&D systems). After 3 days the expressionof CD25 on memory cells (CD45RO⁺ cells) were analyzed by FACS (FIG. 9A).

Similarly, mouse CD4⁺ T cells were isolated from spleen by MACS usingthe “CD4⁺ T cell isolation kit” (Miltenyi) and cultured with 10 ng/mlrmIL-2 (R&D systems) and 10 μg/ml TNFR2 specific antibody or indicatedamount of rmTNF-α (R&D systems). The expression of CD25 on memory cells(CD44⁺CD62L⁻ cells) were analyzed by FACS after 3 days (FIG. 9B).

The percentage of CD25 expressing cells was enhanced in memory cellcultures stimulated with non-blocking TNFR2, in both human and mouse.However, stimulation with blocking antibodies did not increase CD25expression in these cultures but rather decreased this.

In summary, the data in example 4 show that ligand blocking antibodiesare antagonistic as measured by several methods in vitro: inhibition ofNK cell mediated IFN-γ release and activation of CD4⁺ memory cells asmeasured by CD25 expression. The antagonistic ligand blocking antibodiesare shown according to the invention while the agonistic ligandnon-blocking antibodies are included for comparison.

Example 5—Surrogate Ligand Blocking, Antagonistic Anti-Mouse TNFR2 mAbhas In Vivo Anti-Tumor Effect

(See also FIGS. 10-16 and the above description of these figures.)

Therapeutic Effect in Different Tumor Models

To assess the in vivo anti-tumor effect of ligand blocking, antagonisticanti-TNFR2 mAbs a mouse surrogate, called 3F10, was investigated in vivoin different tumor models, using different isotype formats, and alone orin combination with anti-PD-1 as described below.

Mice were bred and maintained in local facilities in accordance withhome office guidelines. Six to eight weeks-old female BalbC and C57/BL6mice were supplied by Taconic (Bomholt, Denmark) and maintained in localanimal facilities. CT26, MC38 and B16.F10 cells (ATCC) were grown inglutamax buffered RPMI, supplemented with 10% FCS. When cells were semiconfluent they were detached with trypsin and resuspended in sterile PBSat 10×10⁶ cells/ml. Mice were s.c. injected with 100 μl cell suspensioncorresponding to 1×10⁶ cells/mouse. 3-8 days after injection dependenton model, mice were treated twice weekly with 10 mg/kg antibody i.p(isotype control, 3-F10 or 5-A05) and as indicated in figures. Tumorswere measured two times/week until they reached a diameter of 15 mm,where after the mice were terminated

The ligand blocking, ant agonistic anti-mouse TNFR2 mAb 3-F10 showtherapeutic anti-tumor effect in three different tumor models (FIG.10-13), with curative effect in more treatment sensitive CT26 (FIG. 10)and tumor growth inhibiting effect in more treatment resistant MC38 andB16 (FIG. 11-13).

The Anti-Tumor Effect of Ligand Blocking, Antagonistic Anti-Mouse TNFR2mAb is Fc:FcγR-Dependent

To assess the importance of Fc-FcγR interaction on the in vivoanti-tumor effect of the ligand blocking, antagonistic anti-TNFR2 mousesurrogate mAb different Fc formats of this antibody was investigated invivo in the CT26 tumor model as described below.

Mice were bred and maintained as described above. CT26 cells (ATCC) weregrown and injected as described above. When tumors reached 3×3 mm, micewere treated twice weekly with 10 mg/kg antibody i.p (isotype control,3-F10 IgG1, 3-F10 IgG2a or 3-F10-N297A (Fc defective). Tumors weremeasured two times/week until they reached a diameter of 15 mm, whereafter the mice were terminated.

The Fc-defective 3-F10-N297A shows minimal or no therapeutic activitycompared to isotype control indicating that Fc-engagement is crucial tothe therapeutic efficacy of this ligand blocking, antagonisticanti-mouse TNFR2 mAb (FIGS. 10 A and B). Both IgG1 and IgG2a formatsshow significant therapeutic efficacy. However, the IgG2a formatpreferentially binding to activating Fcγ-receptors shows superiortherapeutic effect indicative of depletion/phagocytosis of Tregs beingone important mechanism of action of this ligand blocking, antagonisticanti-mouse TNFR2 mAb (FIG. 10 A-B). This is in contrast to anon-blocking, agonistic surrogate antibody 5A05 which shows someactivity in the Fc defective format and shows best activity in themurine IgG1 format, known to preferentially bind the inhibitory FcγR.The ligand-blocking antagonistic antibody (3F10) is in accordance withthe invention and the ligand non-blocking agonistic antibody (5A05) isincluded for reference.

Combinational Effect with Anti-PD-1 mAb

To assess the combinational in vivo anti-tumor effect of ligandblocking, antagonistic anti-TNFR2 mAbs a mouse surrogate (3-F10) withanti-PD-1, the treatment combination was investigated in vivo in theMC38 tumor model as described below.

Mice were bred and maintained as described above. MC38 cells (ATCC) weregrown and injected as described above. Eight days after injection, micewere treated twice weekly with 10 mg/kg antibody i.p (isotype control,anti-mouse PD-1,3-F10 or a combination of anti-mouse P-D-1 and 3-F10)and as indicated in FIG. 11 A-E. Tumors were measured two times/weekuntil they reached a diameter of 15 mm, where after the mice wereterminated.

The anti-mouse PD-1 and the ligand blocking, antagonistic anti-mouseTNFR2 mAb 3-F10 both show tumor growth inhibiting therapeutic effect ineffect in the MC38 model (FIG. 11 A-E). When the anti-PD1 and theantagonistic anti-mouse TNFR2 mAb 3-F10 are combined, tumors are curedin the treatment resistant MC38 (FIG. 11 D-E).

Combinational Effect with Anti-PD-L1 mAb

To assess the combinational in vivo anti-tumor effect of ligand blockingantagonistic anti-TNFR2 mAbs, we further combined the mouse surrogate(3F10) with anti-PD-L1 for treatment in the MC38 tumor model asdescribed below.

Mice were bred and maintained as described above. MC38 cells (obtainedfrom Dr M. Cragg, Southampton University) were grown and injected asdescribed above. Six days after injection, mice were treated twice withisotype control antibody or 3F10 (day 1 and 4), or four consecutive dayswith anti-PD-L1 (clone 10F.9G2, Bioxcell) followed by a fifth injectiontwo days later (in total five injections day 1, 2, 3, 4 and 7), or acombination of both. All antibodies were administered at 10 mg/kg i.p.Tumors were measured with calipers twice weekly until they reached avolume of 2000 mm3, where after the mice were terminated.

The anti-mouse PD-L1 and the ligand blocking, antagonistic anti-mouseTNFR2 mAb 3-F10 both show tumor growth inhibiting therapeutic effect ineffect in the MC38 model (FIG. 12). When the anti-PD-L1 and theantagonistic anti-mouse TNFR2 mAb 3-F10 are combined, the anti-tumoreffect is even further enhanced (FIG. 12).

Immune Cell Modulation In Vivo

To investigate the effects in immune cell in the tumor in vivo, BalbCmice were inoculated with CT26 cells as described above. After thetumors reached approximately 7×7 mm, the mice were treated with 10 mg/kgantibodies administered i.p. as indicated in figures. Mice were treatedat day 1, 4 and 7 and terminated at day 8. Tumors were dissected out,mechanically divided into small pieces and digested using a mixture ofCollagenase 100 μg/ml liberase and 100 μg/ml Dnase in 37° C. for 2×5 minwith Vortex in between. After filtration through a 70 μm filter, thecell suspension was washed (400 g for 10 min) with PBS containing 10%FBS. Thereafter, the cells were resuspended in MACS buffer and stainedwith an antibody panel staining CD45, CD3, CD8, CD4 and CD25 or anantibody panel staining MHCII, F4/80, Ly6C, CD11b and Ly6G. Beforestaining, the cells were blocked for unspecific binding using 100 μg/mlIVIG (purified intravenous immunoglobulins). Cells were analyzed in aFACS Verse. Mouse Tregs were quantified as being CD45⁺CD3⁺CD4⁺CD25⁺ andTAMs as being CD11b⁺Ly6G⁻Ly6C⁻F4/80⁺MHCII⁺. The results are shown inFIG. 14.

As seen in FIG. 14, treatment with the ligand blocking/antagonisticTNFR2 antibody results in an T reg depletion in the tumor. A weakertendency towards increase in CD8⁺ T cell influx is also seen. Together,this results in a much improved CD8+ T cell to T reg ratio (FIG. 14 C).In addition, the antagonistic antibody modulates the myeloid compartmentby reducing the number of tumor associated macrophages (FIG. 14 D).

PBMC-NOG/SCID Model

To confirm the in vivo findings on the depleting activity of the ligandblocking, antagonistic anti-mouse TNFR2 surrogate mAb, we analyzed thedepleting capacity of the ligand blocking, antagonistic anti-human TNFR2mAb 1-H10 in the PBMC-NOG/SCID model in vivo as described below.

Mice were bred and maintained in local facilities in accordance withhome office guidelines. Eight weeks-old female SCID and NOG mice weresupplied by Taconic (Bomholt, Denmark) and maintained in local animalfacilities. For the PBMC-NOG/SCID (primary human xenograft) model, humanPBMCs were isolated using Ficoll Paque PLUS and after washing the cellswere resuspended in sterile PBS at 75×10⁶ cells/ml. NOG mice were i.v.injected with 200 μl cell suspension corresponding to 15×10⁶cells/mouse. 2 weeks after injection, the spleens were isolated andrendered into a single cell suspension. Thereafter, a small sample wastaken to determine the expression of TNFR2 on human T cells by FACS(FIG. 15). This FACS showed that the TNFR2 expression on Tregs and CD8⁺T ceils are very comparable between the human T cells grown andactivated in vivo in the NOG mice, and T cells from human tumors. Themajority of the cells was resuspended in sterile PBS at 50×10⁶ cells/ml.SCID mice were injected i.p. with 200 μl of the suspension correspondingto 10×10⁶ cells/mouse. 1 h later, mice were treated with 10 mg/kg ofeither Yervoy, anti-CD25, 1-H10, 1-H10-N297Q (Fc defective version of1-H10) or isotype control mAb. The intraperitoneal fluid of the mice wascollected after 24 h. Human T cell subsets were identified andquantified by FACS using following markers: CD45, CD3, CD4, CD8, CD25,CD127 (all from BD Biosciences).

1-H10 Treg depleting activity was superior to Yervoy and 1-H10N297Q(FIG. 16), confirming that ligand blocking, antagonistic anti-TNFR2 mAb1-H10 depletes Tregs and that Fc interaction is involved in thisdepletion (FIG. 16 D).

In summary, example 5 shows that:

1. Antagonistic ligand blocking antibodies can have strong anti-tumoreffects across several tumor models

2. This effect can be increased by combining with anti-PD1 antibodies

3. The effect is dependent on engagement of activating FcγRs

4. Treatment with the antagonistic ligand blocking surrogate antibodysignificantly alters the T cell composition in tumors, with an increasedCD8⁺ T cell/Treg ratio, and reduction in tumor associated macrophages.

5. In human tumors, the highest TNFR2 expressing cells are Tregs

6. In a human xenograft model where tumor TNFR2 expression is mimickedon T cells, human Tregs are delated and CD8⁺ T cell levels increased.

7. This Treg deletion is most pronounced if the antibody can engageactivating FcγRs.

Example 6—Antagonistic Ligand-Blocking Antibodies do not Induce LargeAmounts of Proinflammatory Cytokines

(See also FIGS. 17-18 and the above description of these figures.)

Release of large amounts of pro-inflammatory cytokines is one possibleside effect of immune modulatory antibodies used for treatment ofpatients. Hence, we here measured cytokine release induced byantagonistic, ligand blocking antibodies using two different methods.The first is based on antibody stimulation in in vitro cultures, and thesecond is based on xenografting human immune cells to immune deficientmice. For in vitro, the set-up of the culture has been shown to largelyimpact the release of cytokines (Vessillier et al., J Immunol Methods.2015 September; 424: 43-52). To account for differences inmethodologies, three different in vitro culture set-ups were used inaccordance with recent publications.

For the High Density Cell Culture (HDC) Cytokine Release Assays (CRA),PBMCs were cultured at 1×10⁷ cells/ml in serum-free CTL-Test medium(Cell Technology Limited) supplemented with 2 mM glutamine, 1 mMpyruvate, 100 IU/ml of penicillin and streptomycin. 2 ml of cell culturewas plated in a 12-well plate. After 48 h, 10 μg/ml antibody was addedto 1×10⁵ pre-incubated PBMCs in a 96-well flat-bottom plate andincubated for 24 h.

The PBMC Solid Phase (SP) CRA was performed by coating wells of a96-well plate with 1 μg/ml antibody for 1 h. After washing of the platewith PBS, 1×10⁵ PBMCs in 200 μl complete medium were added per well andincubated for 48 h.

The cytokine release was also measured after stimulation of 200 μl ofwhole blood with 5 μg/ml of antibody for 48 h.

At the end of the incubation period, plates were centrifuged and theculture supernatant was taken and stored at −20° C. Concentrations ofIFN-γ, IL-2, IL-4, IL-6, IL-10, IL-8 and TNF-α were measured usingcustom made MSD plates, according to the manufacturer'instructions (MesoScale Discovery, USA).

In summary, the blocking antagonistic antibody did not induce anysignificant cytokine release in any of the in vitro settings. Thepositive control antibodies, Alemtuzumab and OKT3 did induce cytokines,most pronounced of all was IFN-γ, but as seen in FIG. 17, the 1H10antibody did not induce IFN-γ beyond an isotype control antibody. Noother cytokine was elevated by 1H10 (data not shown).

PBMC-NOG Tolerability Model

To investigate the tolerability of the ligand blocking, antagonisticanti-human TNFR2 mAb 1-H10, we analyzed the in vivo cytokine release inthe PBMC-NOG model as described below.

Mice were bred and maintained in local facilities in accordance withhome office guidelines. Eight weeks-old female NOG mice were supplied byTaconic (Bomholt, Denmark) and maintained in local animal facilities.For the PBMC-NOG (primary human xenograft) model, human PBMCs wereisolated using Ficoll Paque PLUS and after washing the cells wereresuspended in sterile PBS at 125×10⁶ cells/ml. NOG mice were i.v.injected with 200 μl cell suspension corresponding to 25×10⁶cells/mouse. 2 weeks after injection, blood samples were taken toanalyze the level of “humanization” meaning the amount of human cells inthe blood of the NOG mice. The blood was composed of approx. 40% humanT-cells and the mice were considered humanized. The mice were thentreated with 10 μg of either Yervoy, anti-CD3 (OKT-3), 1-H10, or isotypecontrol mAb. Body temperature was measured prior to antibody injectionand at 1 h post injection FIG. 18 A. As seen in FIG. 18 A, the positivecontrol antibody OKT3 induced dramatic lowering of body temperature aspreviously published, and in accordance with the toxicity seen in theclinic with this antibody. In contrast, 1-H10 did not show any effect onbody temperature. Five hours post injection of antibodies theexperiments were terminated and blood was collected for analysis ofcytokine release (MSD). The cytokines measured were human IFN-γ, TNF-α,IL-6 and IL1β. Of these, IFN-γ and TNF-α were quantified in high enoughlevels to be reliable. As seen in FIGS. 18 B and C, the positive controlantibody OKT3 induced both significant IFN-γ and TNF-α release (inaccordance with the toxicity seen in the clinic with this antibody)whereas 1H10 treated mice had no significant IFN-γ release. However,there was a tendency towards increase in TNF-α release, although notsignificant and as dramatic as for OKT3.

In summary, example 6 shows that the TNFR2 ligand blocking antibodies,here exemplified with the antibody called 1-H10, do not inducesubstantial levels of cytokine release as measured by several previouslypublished methods. Since cytokine release is a limiting factor forclinical development of several immunomodulatory antibodies, thisindicates an acceptable safety profile in this regard.

Example 7—Epitopes of Generated TNFR2 Targeting Antibodies DomainConstruct Knock-Outs

In a first set of experiments, DNA constructs encoding differentvariants of TNFR2, missing one or more of the 4 extracellular domains,described in table 10, were used. In a second set of experiments, DNAconstructs encoding variants of TNFR2 where different parts of domain 3were exchanged with the corresponding murine part, as described in table11, were used. The latter is possible since none of the antibodies iscross-reactive to murine TNFR. In both cases, the constructs werepurchased from GeneArt (ThermoFisher). The constructs were cloned intoan expression vector, containing the CMV-promotor and the OriP origin ofplasmid replication, and transiently expressed in suspension adaptedHEK293-EBNA cells.

TABLE 10 TNFR2 constructs used for transfection where one or severaldomains have been deleted. Construct Description hTNFR2 wild type, fulllength human TNFR2 (uniprot #P20333) hTNFR2-Δ1 hTNFR2 with domainTNFR-Cys 1 (aa 39-76) deleted hTNFR2-Δ2 hTNFR2 with domain TNFR-Cys 2(aa 77-118) deleted hTNFR2-Δ3 hTNFR2 with domain TNFR-Cys 3 (aa 119-162)deleted hTNFR2-Δ4 hTNFR2 with domain TNFR-Cys 4 (aa 163-201) deletedhTNFR2-Δ1 + hTNFR2 with domain TNFR-Cys 1 and 3 (aa 39-76 3 and 119-162)deleted hTNFR2-Δ2 + hTNFR2 with domain TNFR-Cys 2 and 4 (aa 77-118 4 and163-201) deleted

TABLE 11 TNFR2 constructs used for transfection various parts of domain3 have been exchanged for the corresponding murine sequence. ConstructDescription hTNFR2 wild type, full length human TNFR2 (uniprot #P20333)mTNFR2 wild type, full length murine TNFR2 (uniprot #P25119) hTNFR2-m1hTNFR2 with aa 119-132 replaced by aa 120-133 from mTNFR2 hTNFR2-m2hTNFR2 with aa 134-144 replaced by aa 135-146 from mTNFR2 hTNFR2-m3hTNFR2 with aa 151-160 replaced by aa 153-162 from mTNFR2 hTNFR2-m4hTNFR2 with aa 130-144 replaced by aa 131-146 from mTNFR2

Flow Cytometry Based Binding Analysis

HEK-293-E cells were transfected with the respective cDNA plasmids ofTNFR2 variants using Lipofectamin 2000.48 h after transfection, cellswere harvested and stained with the indicated antibodies for 30 minutes.After 2 washing steps with PBS, surface bound antibodies were stainedwith a secondary anti-IgG coupled to APC. Prior to flow cytometryanalysis on BD-Verse flow cytometer, cells were washed and stained forlive/dead.

Flow cytometry based binding experiments of transfected HEK 293 cellsdearly showed, that domain 1 and domain 2 either does not affect thebinding (domain 1), or only marginally affects binding (domain 2), ofany of the antibodies to these cells. As a positive control a polyclonalanti-human TNFR2 antibody was used. The positive control antibody showedhigh binding to all tested constructs, whereas the negative antibodyshowed no binding (FIG. 19). All tested antibodies showed a completeloss of binding to TNFR2 lacking domain 3. Similarly, most antibodiescould not bind to TNFR2 if domain 4 was missing. All antagonisticantibodies (1H10, 4H02 and 5B08) showed drastically reduced binding toTNFR2 Δ4 of more than 50% compared to binding to TNFR2 Δ1 and TNFR2 Δ2.Similarly, removing two domains from TNFR2 clearly showed that the lackof domain 3 or 4 severely abrogated binding of all tested antibodies toTNFR2, with the possible exception of agonistic antibody 1F06, while thelack of domain 4 abolished binding of agonistic antibodies and reducedthe binding of the antagonistic antibodies significantly. (FIGS. 19 Eand F).

Binding to Mouse-Human Chimeric TNFR2

To further narrow down the binding site and define the epitopes, partsof the human TNFR2 domain 3 were replaced by the corresponding mousesequence. Since all antibodies shows very little cross-reactivity tomouse TNFR2, a loss of binding to certain constructs would allowrefining the binding epitope. FIG. 20 displays the different mouse-humanchimeric TNFR2 constructs. Four different replacements were made,exchanging either 14 (m1), 12 (m2), 10 (m3) or 16 (m4) amino acids fromthe human sequence with the corresponding mouse sequence. The otherthree domains (1, 2, 4) contain exclusively human sequences.

These constructs (TNFR2 domains 1-4 with mutations in 3) were thentransfected into HEK293 cells and antibodies were tested for bindingusing a flow cytometry approach. As positive controls, polyclonalantibodies against mouse TNFR2 as well as against human TNFR2 were used.As expected, due to sequence similarity, both polyclonal controlantibodies showed significant cross-reactivity and recognized both,human and mouse TNFR2. Obviously, best signals were achieved whenmatching the antibodies to its intended target.

Our monoclonal antibodies showed strong binding to human TNFR2, but noor only very little binding to mouse TNFR2 (FIG. 21 left panels).Similar binding with very little reduction was observed for all clonesto the hTNFR2 m1 construct with mutations in aa 119-132, indicating,that none of the antibodies bind to an epitope within that region.However, mutations in aa 134-144 (hTNFR2 m2 construct) abrogated bindingcompletely for half of the tested antibodies, corresponding to theantagonistic blocking antibodies 1-H10, 4-H02 and 5-B08, indicating thatthe antibodies bind at least partially within this region. The 1-G10 isa partial blocker also strongly affected by this replacement.Noteworthy, the agonistic antibodies (1-F02, 1-F06 and 4-E08) retainedbinding using construct 2, strongly suggesting a different epitopecompared to the antagonistic antibodies. Interestingly, all antibodieslost binding to the hTNFR2 m3 construct with mutations in aa 151-160.This indicates, that all antibodies, both agonists and antagonists, haveat least a partial epitope within that a sequence. Testing a slightlylarger construct hTNFR2 m4 with mutations in aa 130-144 showed similarbinding as with construct hTNFR2 m2.

Conclusions Binding Epitopes

Grouping the antibodies into their functionally role, the agonisticantibodies (1-F02, 1-F06 and 4-E08), seems to bind a very distalC-terminal part of domain 3 encompassing aa 151-160 and likely extend toa larger part of domain 4, whereas the epitope for the antagonists(1-H10, 5-B08 and 4-H02) are shifted more towards the center of domain3, encompassing aa 134-160 and probably covers a smaller part of domain4. However, despite this, their epitopes seem to overlap to some extent.

None of the antibodies bind to the N-terminal part of domain 3, aa119-134. Binding sites to domain 4 is quite likely for all antibodies,but has not been identified completely.

1. An antagonistic antibody molecule that specifically binds to TNFR2 ona target cell and thereby blocks TNF-α binding to TNFR2 and blocks TNFR2signaling, and wherein the antibody molecule also binds to an Fcγreceptor via its Fc region.
 2. An antibody molecule according to claim1, wherein the antibody binds to with higher affinity to activating Fcγreceptors than to inhibitory Fcγ receptors.
 3. An antibody moleculeaccording to claim 1, wherein the binding of the antibody molecule toTNFR2 results in change in numbers and/or frequency of TNFR2 expressingcells in diseased tissue.
 4. An antibody molecule according to claim 1,wherein the binding of the antibody molecule to TNFR2 results ininfiltration of T-cells and/or myeloid cells into diseased tissue and/ora change in composition of T-cells and/or myeloid cells in diseasedtissue.
 5. An antibody molecule according to claim 1, wherein theantibody molecule is selected from the group consisting of: a full-sizeantibody, a chimeric antibody, a single chain antibody, and anantigen-binding fragment thereof retaining the ability to bind an Fcreceptor via its Fc region.
 6. An antibody molecule according to claim1, which binds to human TNFR2 (hTNFR2) and/or to cynomolgus monkey TNFR2(cmTNFR2).
 7. An antibody molecule according to claim 1, wherein theantibody molecule is selected from the group consisting of a human IgGantibody molecule, a humanized IgG antibody molecule, and an IgGantibody molecule of human origin.
 8. An antibody molecule according toclaim 7, wherein the antibody molecule is a human IgG1 antibody.
 9. Anantibody molecule according to claim 7, wherein the antibody moleculehas been engineered for improved binding to activating Fc gammareceptors.
 10. An antibody molecule according to claim 1, wherein theantibody molecule is a monoclonal antibody.
 11. An antibody moleculeaccording to claim 1, wherein the antibody molecule does not bindspecifically to an epitope comprising or consisting of the sequenceKCSPG.
 12. An antibody molecule according to claim 1, wherein theantibody molecule is selected from the group consisting of antibodymolecules comprising 1-6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1,VL-CDR2 and VL-CDR3, wherein VH-CDR1, if present, is selected from thegroup consisting of SEQ. ID. NOs: 1, 9 and 17; wherein VH-CDR2, ifpresent, is selected from the group consisting of SEQ. ID. NOs: 2, 10and 18; wherein VH-CDR3, if present, is selected from the groupconsisting of SEQ. ID. NOs: 3, 11 and 19; wherein VL-CDR1, if present,is selected from the group consisting of SEQ. ID. NOs: 4, 12 and 20;wherein VL-CDR2, if present, is selected from the group consisting ofSEQ. ID. NOs: 5, 13 and 21; and wherein VL-CDR3, if present, is selectedfrom the group consisting of SEQ. ID. NOs: 6, 14 and
 22. 13. An antibodymolecule according to claim 1, wherein the antibody molecule comprises avariable heavy chain (VH) comprising the following CDRs (i) SEQ. ID. NO:1, SEQ. ID. NO: 2 and SEQ. ID. NO: 3; or (ii) SEQ. ID. NO: 9, SEQ. ID.NO: 10 and SEQ. ID. NO: 11; or (iii) SEQ. ID. NO: 17, SEQ. ID. NO: 18and SEQ. ID. NO: 19; and/or wherein the antibody molecule comprises avariable light chain (VL) comprising the following CDRs: (i) SEQ. ID.NO: 4, SEQ. ID. NO: 5 and SEQ. ID. NO: 6; or (ii) SEQ. ID. NO: 12, SEQ.ID. NO: 13 and SEQ. ID. NO: 14; or (iii) SEQ. ID. NO: 20, SEQ. ID. NO:21 and SEQ. ID. NO:
 22. 14. An antibody molecule according to claim 1,wherein the antibody molecule comprises a variable heavy chain (VH)amino acid sequence selected from the group consisting of SEQ. ID. NOs7, 15 and 23; and/or wherein the antibody molecule comprises a variablelight chain (VL) amino acid sequence selected from the group consistingof SEQ. ID. NOs: 8, 16 and
 24. 15. An antibody molecule according toclaim 1 wherein the antibody molecule is an antibody molecule that iscapable of competing for binding to TNFR2 with an antibody moleculeselected from the group consisting of antibody molecules comprising 1-6of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3,wherein VH-CDR1, if present, is selected from the group consisting ofSEQ. ID. NOs: 1, 9 and 17; wherein VH-CDR2, if present, is selected fromthe group consisting of SEQ. ID. NOs: 2, 10 and 18; wherein VH-CDR3, ifpresent, is selected from the group consisting of SEQ. ID. NOs: 3, 11and 19; wherein VL-CDR1, if present, is selected from the groupconsisting of SEQ. ID. NOs: 4, 12 and 20; wherein VL-CDR2, if present,is selected from the group consisting of SEQ. ID. NOs: 5, 13 and 21; andwherein VL-CDR3, if present, is selected from the group consisting ofSEQ. ID. NOs: 6, 14 and
 22. 16. An isolated nucleotide sequence encodingan antibody molecule as defined in claim
 1. 17. A plasmid comprising anucleotide sequence as defined in claim
 16. 18. A virus comprising anucleotide sequence as defined in claim
 16. 19. A virus according toclaim 18, further comprising a nucleotide sequence encoding an antibodymolecule that specifically binds to a check-point inhibitor.
 20. A cellcomprising a nucleotide sequence as defined in claim
 16. 21. A method oftreating a disease comprising administering to a patient an antibodymolecule as defined in claim
 1. 22. A method of treating cancer or aninfection caused by an intracellular pathogen comprising administeringto a patient an antibody molecule as defined in claim
 1. 23. A methodaccording to claim 22, wherein the patient to be treated is a patienthaving high TNFR2 expression in diseased tissue.
 24. A method oftreating cancer comprising administering to a patient an antibodymolecule as defined in claim 1, wherein said antibody is administered incombination with: an antibody molecule that specifically binds to acheck-point inhibitor; a nucleotide sequence encoding an antibodymolecule that specifically binds to a checkpoint inhibitor; a plasmidcomprising a nucleotide sequence encoding an antibody molecule thatspecifically binds to a check-point inhibitor, and/or a cell comprisinga nucleotide sequence encoding an antibody molecule that specificallybinds to a check-point inhibitor, a plasmid comprising a nucleotidesequence encoding an antibody molecule that specifically binds to acheck-point inhibitor or a virus comprising a nucleotide sequenceencoding an antibody molecule that specifically binds to a check-pointinhibitor. 25-27. (canceled)
 28. A pharmaceutical composition comprisingor consisting of an antibody molecule as defined in claim 1, and furthercomprising a pharmaceutically acceptable diluent, carrier, vehicleand/or excipient.
 29. (canceled)
 30. A method of treating cancer,comprising administering a pharmaceutical composition according to claim28 in combination with a second pharmaceutical composition comprising:an antibody molecule that specifically binds to a check-point inhibitor;a nucleotide sequence encoding an antibody molecule that specificallybinds to a checkpoint inhibitor; a plasmid comprising a nucleotidesequence encoding an antibody molecule that specifically binds to acheck-point inhibitor, and/or a cell comprising a nucleotide sequenceencoding an antibody molecule that specifically binds to a check-pointinhibitor, a plasmid comprising a nucleotide sequence encoding anantibody molecule that specifically binds to a check-point inhibitor ora virus comprising a nucleotide sequence encoding an antibody moleculethat specifically binds to a check-point inhibitor.
 31. (canceled)
 32. Amethod according to claim 22, wherein the patient is a patient havinghigh TNFR2 expression in diseased tissue.
 33. A method for treatment ofcancer according to claim 30, wherein also a therapeutically effectiveamount of an antibody molecule that specifically binds to a check-pointinhibitor is administered to the patient.
 34. A method according toclaim 24, wherein the check-point inhibitor is PD-1.
 35. A methodaccording to claim 24, wherein the check-point inhibitor is PD-L1.
 36. Amethod according to claim 22, wherein the cancer is a solid cancer.